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STUDIES ON THE CHEMICAL CONSTITUENTS OF HEDERA
NEPALENSIS K. KOCH AND CORNUS MACROPHYLLA WALL. EX
ROXB
Ph.D. Thesis
By
ASHFAQ AHMAD KHAN
INSTITUTE OF CHEMICAL SCIENCES
UNIVERSITY OF PESHAWAR,
PESHAWAR, PAKISTAN
FEBRUARY, 2014
STUDIES ON THE CHEMICAL CONSTITUENTS OF HEDERA
NEPALENSIS K. KOCH AND CORNUS MACROPHYLLA WALL. EX
ROXB
By
ASHFAQ AHMAD KHAN
A dissertation
Submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Chemistry
INSTITUTE OF CHEMICAL SCIENCES
UNIVERSITY OF PESHAWAR,
PESHAWAR, PAKISTAN
FEBRUARY, 2014
“Start from the name of ALLAH, the most beneficent, the mostmerciful”
DEDICATED TO
MY
PARENTS AND FAMILY MEMBERS
CONTENTS
Acknowledgments……………………………………………………………………………i
Abstract………………………………………………………………………………............iii
List of the Abbreviations……………………………………………………………………..v
List of the Tables………………………………………………………………………..........vii
List of the Figures…………………………………………………………………………….ix
List of the Schemes……………………………………………………………………...........x
Chapter 1 Part A
1.0. General Introduction……………………………………………………………...1
Chapter 2 Plant Introduction…………………….
2.1. Plant Introduction …………………………………………………………………..........7
2.1.1. Genus Hedera…………………………………………………………….............7
2.1.2. Hedera nepalensis K. Koch………………………………………………………7
2.1.3. Chemical constituents of the genus Hedera…………………………………………...8
2.1.4. Structures of selected compounds reported from the genus Hedera.....................12
2.1.5. Medicinal and pharmacological properties of the genus Hedera…………….........20
Chapter 3 Results and Discussion …………………
3.1. Present work..................................................................................................................22
3.2. New compound isolated from H. nepalensis................................................................22
3.3. Hitherto unreported compounds from H. nepalensis.....................................................23
3.2.1 Hepalensiside A (1) .........................................................................................25
3.3.1. Colchiside A (2)………………………………………………………………27
3.3.2. Pastuchoside A (3)……………........………………………………………….28
3.3.3. Helixoside A (4)……………………………………………………………….29
3.3.4. Kizuta saponin K12 (5)……………………………………………….……….30
3.4. Biological studies……………………………………………………………………..30
3.4.1. Anti-bacterial assay………………………………………………………….30
3.4.2. Anti-fungal assay……………………………………………………………31
3.4.3. Phytotoxicity assay………………………………………………………….31
3.4.4. Insecticidal assay……………………………………………………………31
3.4.5. Brine shrimp (Artemia salina) lethality bioassay…………………………..32
Chapter 4 Experimental………………………….
4.1. General experimental……………………………………………………………….33
4.2. Plant Material……………………………………………………………………….33
4.3. Extraction and isolation…………………………………………………………….34
4.4. Characterization of chemical constituents…………………………………………36
Chapter 5 Plant Introduction…………… Part B
5.1. Plant Introduction ………………………….............................................................61
5.1.1. Genus Cornus…………………………....................................................................61
5.1.2. Cornus macrophylla Wall…………….....................................................................61
5.1.3.Chemical constituents of the genus Cornus ………………………….........................62
5.1.4. Structures of some selected compounds reported from the genus Cornus...............67
5.1.5. Medicinal and pharmacological properties of the genus Cornus…………………..74
Chapter 6 Results and Discussion ………………………….
6.1. Present work……………………………………………………………………..….77
6.2. New compound isolated from Cornus macrophylla………………………………..77
6.2.1 Macrophyllanin A (6)……………………………………………………...82
6.2.2 Macrophyllanin B (7)……………………………………………………....84
6.2.3 Macrophyllanin C (8)………………………………………………………86
6.2.4 Macrophyllanin D (9)……………………………………………………....88
6.3. Hitherto unreported compounds from Cornus macrophylla…………………….....79
6.3.1. Kaempferol (10) ……………………………….............................................90
6.3.2. Taraxasterol (11) ………………………………............................................90
6.3.3. 3β-Hydroxy-18α-olean-28-19β-olide (12)…………………………………91
6.4. Reported compounds from Cornus macrophylla…………………………………...80
6.4.1. Betulinic acid (13) ………………………………..........................................92
6.4.2. Betulin (14) ………………………………....................................................93
6.4.3. Stigmasterol (15) ………………………………............................................94
6.4.4. Lupeol (16) ……………………………….....................................................95
6.4.5. Oleanolic acid (17) ……………………………….........................................95
6.5. Biological studies ………………………………........................................................96
6.5.1 Anti-bacterial assay ………………………………............................................96
6.5.2 Anti-fungal assay.………………………………................................................96
6.5.3 Phytotoxicity assay .………………………………............................................97
6.5.4 Insecticidal assay………………………….......................................................97
6.5.5. Brine shrimp (Artemia salina) lethality bioassay.…………………………...97
Chapter 7 Experimental………………………….
7.1. General experimental…………………………………………………………….....101
7.2. Plant Material…………………………………………………………………….....101
7.3. Extraction and isolation ……………………………………………………………101
7.3.1. Fraction of ethyl acetate phase ……………………………………………....101
7.4. Characterization of chemical constituents………………………………………......105
References ………………………………………………………………………………….132
List of Publication …………………………………………………………………………140
i
Acknowledgments
At first I want to express my unfeigned thanks and praise to Almighty Allah, Who in his great
mercy and benevolences has enabled me to carry out and complete this Ph.D. research work.
I would like to express my special appreciation and thanks to my advisor Professor Dr. Ghias
Uddin, you have been a tremendous mentor for me. I would like to thank you for encouraging
my research and for allowing me to grow as a research scientist. Your advice on both research
as well as on my career have been priceless.
My special gratitude and thanks to my co-supervisor Prof. Dr. Bina Shaheen Siddiqui (HEJ
Research institute of Chemistry, University of Karachi) for her guidance.
I would like to give special thanks to my supervisors Dr. Valerie. A. Ferro and Prof Dr.
Alexander Irvine Gray (SIPBS, University of Strathclyde, Glasgow UK.) for their unflagging
support, guidance and provided me a unique opportunity to work in their research center.
I am particularly indebted to Prof. Mingkui Wang (Chengdu Institute of Biology, Chinese
Academy of Sciences.) who guided me during the course of my Ph.D. His everlasting
encouragements, supervision and generous help facilitate my research work.
I feel great pleasure in expressing my special thanks to Prof. Dr. Yousaf Iqbal (Director,
Institute of Chemical Sciences, University of Peshawar) and all the faculty members of ICS
for their support and co-operation.
My special gratitude and thanks to Prof. Dr. Abdur Rashid (Ex-chairman Botany Department,
University of Peshawar) and Abdul Majid (Lecturer Department of Botany, Hazara
University, Mansehra) for the identification of my plants species on such a short notice.
ii
I want to sincerely acknowledge all my friends and colleagues, Dr. Waliullah, Dr. Alauddin,
Dr. Abdul Latif, Dr. Hamid Hussain, Dr. Muhammad Alamzeb, Anwar Sadat, Muhammad
Alam, Saqib Ali, Mamoon-Ur-Rashid and Abdur Rauf for their encouragement, support and
help all the time.
In the last but not least, I am truly indebted to my parents and family members whose prayers
and support have enabled me to complete the Ph.D. research work. I would like to thank my
parents for allowing me to realize my own potential. All the support they have provided me
over the years was the greatest gift.
I am also greatful to the Higher Education Commission of Pakistan for research funds.
Ashfaq Ahmad Khan
iii
Abstract
The research work carried out for the doctoral dissertation is mainly focused on
bioassayguided isolation, characterization and structure determination of pure chemical
constituents of H. nepalensis K. Koch (Part-A) and C. macrophylla Wall (Part-B).
The structures of the pure chemical constituents were elucidated by advanced spectroscopic
methods.
Part A
Chemical explorations of H. nepalensis were described in part A; resulted in the isolation of
one new triterpenoid saponin; hepalensiside A (1) and four hitherto unreported triterpenoid
saponins; colchiside A (2), pastuchoside A (3), helixoside A (4) and kizuta saponin K12 (5)
from the leaves of H. nepalensis. Various fractions and extract of H. nepalensis were also
evaluated for different bioassays. As a result semi-purified fractions exhibited significant anti-
bacterial activities against P. mirabilis at conc. of 12 μg/ml, against B. cereus 13 and E. coli
11 μg/ml, while the methanolic extract of the H. nepalensis was found to be cytotoxic.
iv
Part B
Part B described the phytochemical studies of C. macrophylla, affording four new
chemical constituents; macrophyllanin A (6), macrophyllanin B (7), macrophyllanin C
(8) and acrophyllanin D (9), together with three hitherto unreported compounds;
kaempferol (10), taraxasterol (11) 3β- hydroxy-18α-olean-28-19β-olide (12) and five
known compounds; betulinic acid (13), betulin (14), stigmasterol (15), lupeol (16) and
oleanolic acid (17). Semi-purified fractions were also evaluated for biological assays, as
a result some fractions were found to be active against B. cereus and Lemna minor (plant)
and were also showed cytotoxic effect.
6 7
.
8 9
v
List of Abbreviations
13C- NMR Carbon-13 Nuclear Magnetic Resonance Spectroscopy
COSY Correlation Spectroscopy
DEPT Distortion less Enhancement by Polarization Transfer
1D NMR One Dimensional Nuclear Magnetic Resonance Spectroscopy
2D NMR Two Dimensional Nuclear Magnetic Resonance Spectroscopy
ESI-MS Electrospray Ionization Mass Spectroscopy
EIMS Electron Mass Impact Spectroscopy
ESI Electrospray Ionization
FAB-MS Fast Atomic Bombardment Mass Spectrometry
Hz Hertz
HMBC Heteronuclear Multiple Bond Correlation
HMQC Heteronuclear Multiple Quantum Coherence
1H-NMR Proton Nuclear Magnetic Resonance Spectroscopy
HR-ESI-MS High Resolution Electrospray Ionization Mass Spectroscopy
HSQC Heteronuclear Single Quantum Coherence
IR Infrared spectroscopy
J Coupling Constant
MS Mass Spectroscopy
MIC Minimum Inhibitory Concentration
m/z Mass to Charge ratio
mp Melting Point
NOESY Nuclear Overhauser Effect Spectroscopy
vi
NMR Nuclear Magnetic Resonance
ppm Parts Per Million
st Stretching
UV-Vis Ultraviolet-Visible Spectroscopy
ῡ Wave number
vii
List of Tables
Table. 2.1. Chemical constituents of the genus Hedera…………………………... 9
Table. 4.1. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 1 in C5D5N…… 37
Table. 4.2. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 2 in C5D5N…… 40
Table. 4.3. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 3 in C5D5N……... 43
Table. 4.4. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 3 for sugar
moieties at position C-28 ……………………………………………… 45
Table. 4.5. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 4 in C5D5N…… 48
Table. 4.6. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 4 for sugar
moieties at position C-28 ……………………………………………... 50
Table. 4.7. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 5 in C5D5N…… 52
Table. 4.8. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 5 for sugar
moieties of 5 at position C-28 ……………………………………….. 54
Table. 4.9. Anti-bacterial activity of H. nepalensis………………………………….. 55
Table. 4.10. Anti-fungal profile of H. nepalensis……………………………………... 56
Table. 4.11 Phytotoxicity assay of H. nepalensis……………………………………... 57
Table. 4.12. Insecticidal assay H. nepalensis………………………………….... 59
Table.4.13. Brine Shrimp lethality bioassay of H. nepalensis…………………… 60
Table. 5.1. Chemical constituents of the Genus Cornus……………………………… 64
Table. 7.1. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 6 in CDCl3…….. 105
viii
Table. 7.2. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 7 in CDCl3………. 107
Table. 7.3. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 8 in CDCl3…… 109
Table. 7.4. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 9 in CDCl3……. 111
Table. 7.5. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 10 in CDCl3……... 113
Table. 7.6. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 11 in CDCl3……... 115
Table. 7.7. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 12 in CDCl3……... 117
Table. 7.8. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 13 in CDCl3……. 119
Table. 7.9. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 14 in CDCl3……... 120
Table. 7.10. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 15 in CDCl3…… 122
Table. 7.11. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 16 in CDCl3…… 124
Table. 7.12. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 17 in CDCl3…… 126
Table. 7.13. Anti-bacterial activity of C. macrophylla……………………………….. 128
Table. 7.14. Anti-fungal assay activity of C. macrophylla………………………... 128
Table. 7.15. Phytotoxicity assay of C. macrophylla ………………………………… 129
Table. 7.16. Insecticidal activity of C. macrophylla………………………………….. 130
Table. 7.17. Brine Shrimp activity of C. macrophylla……………………………….. 131
ix
List of Figures
Figure. 3.1. Important HMBC (H→C) correlations of compound 1………….. 27
Figure. 6.1. Key HMBC correlations of 6…………………………………….. 83
Figure. 6.2. Key HMBC correlations of 7…………………………………….. 85
Figure. 6.3. Key HMBC correlations of 8…………………………………….. 87
Figure. 6.3. Key HMBC correlations of 9…………………………………….. 89
x
List of Schemes
Scheme 4.1. Extraction and isolation from H. nepalensis K. Koch……………. 37
Scheme 7.1. Extraction and isolation from C. macrophylla stem……………… 105
Chapter 1
General Introduction
Chapter 1 General introduction
1
1.0. General introduction
Human being is associated with the therapeutic plants from the beginning of the
human civilization. Plants are used as a source of drugs in all most every part of the world.
Long life, everlasting beauty and perfect health has quest for human being. Man enabled to
distinguish facts from imaginary tales through such ingenious expeditions based on his
dreams and on his practical experiences. It moved to the new group as folk medicines and as
the time passed developed into a field of modern medical science. The hard work of human
being describes the history of medication from the primal endeavors of ancient human being
to the modern advanced age of medical sciences and further be divided into the subsequent
periods. First period expanded over Chinese, Indian, Egyptian, Assyrian, Sumerian and
Babylonian civilizations followed by Arabic, Persian, Greek, Roman and at the end the
modern era. The Greeks originated the modern medicines. The knowledge of medicines
from the Greece was gradually shifted to Romans and then to Arabs. After the advancement
in the field of medical science with the Indian and Chinese medicines, it was transformed to
the current Europe. It was commenced by the Muslim rulers in India and combing together
with the indigenous Ayurvedic medicine, which is called as Eastern or Unani medicine.1, 2
The earliest ideas about the use of medicinal plants were recorded by Rigveda (4500-1600
BC) and Ayurveda (2500-600 BC). Numerous groups of herbs in several sets was searched
and explained by Chopra and his research groups. The use of medicinal plants during
Buddhist period was enhanced and substantial attention was given in a scientific mode for
cultivation of such plants.3
Chapter 1 General introduction
2
Among Chinese Shen Nung accumulated a Pharmacopoeia around 2735 BC on
medicinal plants called as Pen Tsao which consisted of forty volumes and many
therapeutic preparations and literature record of medicinal values of some drugs like Ma
Huang and Chang Sheng extracted from Ephedra sinica and Dichroa febrifuga plants.
Another great work compiled by Li Shizhen in the form of Ben Cao Gang Mu is serving as
a reference book for practicing, teaching and supervision in Chinese therapeutic research.
However majority of the literature concerning Chinese medicine originated from Nei
Ching.4, 5
Numerous medicinal plants have been introduced by Egyptian for the treatment of
human diseases in Ebers Papyrus about 1550 BC. The Assyrian and Babylonian fields of
medicine developed at about 650 BC. In 460 BC Hippocrates, the developer of medicines
put forward the foundation of pharmacy. They illustrated and named about 400 samples
having medicinal valve.
Among Romans, Galen the developer of Roman pharmacology wrote about thirty
books on pharmacology and investigated preparations known as galenicals from many
medicinal plants.
The Muslims during the early and late middle ages made great contribution to the
field of medical science. During the excellent era of Muslims superpower, Arabs took great
interest in the field of herbal medicines and they translated the work to his own language
from almost all the famous civilizations. Abu Bakr Mohammad Bin Zakaria Al-Razi
(Rhazes) the renowned Arab scholar wrote about 250 phenomenal books. Most part of his
work on medicines was illustrated in Alhavi Kabeer (Continens of Rhazes). Kitab-al-
Mansoori is one of his evident books in which he briefly discussed
Chapter 1 General introduction
3
the Greek-Arab system of medication. For the first time in the history Rhazes described
opium as anesthetic agent. Ali Ibn-e-Rabban Tabari wrote a book ‘Firdous Al-Hikmat’
which consists of seven volumes during the period 833-870 AD. The most famous
philosopher, physician, mathematician and astronomer of the Muslims era Bu-Ali Ibn-e-Sina
(908-1037 AD) known as Avicenna in Europe has written 760 herbal drugs in his book
entitled ‘Qanun fi Al-Tibb’ (The Cannon of Medicine) which was an authentic book of
medicines up to 17th century AD. Al-Idrisi another well-known scientist of the Muslim era
(1099-1166 AD) has done a lot of work in the field of medicinal plants therapy and has
written Kitab Al-Jami-ul-sifat Ashat Al-Nabatat.2 F.W. Serturner (1783-1810) in the first
decade of 19th century, isolated morphine from the dried leaf of Papaver sominiferum L
(opium) that opened a new authentic way for the search of valuable herbal drugs from the
natural resources. Pelletier and Cavantou later on isolated cocaine, nicotine, papaverine,
quinine, strychnine from the plants. In 1870 Hoffmann for the first time identified the
structure of coniine and after that it was synthesized by Ladenburg in 1886. Due to their
physiological importance and complicated chemistry these compounds are considered to be
the first of the real pure natural chemical constituents. Several important findings in the field
of medicine can be attributed to the isolation of potent compounds from the natural
sources.6 Different biologically active compounds are present in plants like, glycosides
(saponin glycoside, cardioactive glycoside, anthraquinone glycosides), volatile oils
(peppermint, clove, cardamom oil), alkaloids (morphine, cocaine, atropine), resins, gums
and mucilages. Several important findings in the field of medicine can be accredited to the
isolation of potent compounds from natural sources.7 Atropine, morphine, digoxin, reserpine
and quinine, are some of the examples of plants drugs still used in the modern day
Chapter 1 General introduction
4
therapeutics. Although majority of the plants have been studied for their medicinal
properties but still the numbers of plants that have not been investigated for their
biologically active components is very long.8 It has been reported that the herbal drugs are
less toxic and free from the more side effects than the synthetic drugs so for the industrial
development there is a great potential for the traditional herbal remedies plants extracts for
the treatment and prevention of disease.9 Even though plants have played significant role
from the early ages of many human diseases as basis for the treatment, one wonders that
only two percent plant species have been subjected to pharmaco-chemical investigations. In
one of the reports of WHO, it is recorded that due the convenient availability and socio-
cultural environment of the conventional medicines, more than half of the world population
is still dependent on these types of medicines.10-17 Synthetic drugs such as salvarasan by
Chakravarthi 18 and the use of the chemotherapy in the early decades of 20th century greatly
decreased the value of herbal drugs and increased the concern of people towards synthetic
drugs. Isolation of antibiotics from the plants and the importance of some of the constituents
of medicinal plants for the treatment against cardiovascular diseases and many types of
cancer, which has been an extensive recovery of attention in the field of natural products.
Although all herbal medicines are not useful as claimed, even though they have provided us
massive field for development and investigation. It is the medicinal agents isolated from the
plants which are the starting materials of many active synthetic and semi synthetic drugs.
The task which needs to be accepted is to validate the clinical values of prescribed herbal
medicines. This challenge requires great determination, persistence and scientific
knowledge. In all over the world, the interest of people developed in the chemical and
Chapter 1 General introduction
5
pharmacological valuation of medicinal plants is growing particularly in tropical and sub-
tropical regions.
The work carried out for doctoral dissertation in view of the facts discussed consists
of isolation, structural determination of chemical constituents and studies on the biological
activities of H. nepalensis K. Koch (Part-A) and C. macrophylla Wall (Part-B).
Chapter 2
Plant Introduction
Chapter 2 Plant introduction (Part-A)
6
Hedera nepalensis K. Koch
Chapter 2 Plant introduction (Part-A)
7
2.1. Plant Introduction
Plant description
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Apiales
Family: Araliaceae
Genus: Hedera
Specie: H. nepalensis
Binomial name: Hedera nepalensis K. Koch
2.1.1. Genus Hedera
Genus Hedera belongs to the family Araliaceae consists of 70 genera and 700 species
mainly habitating in Europe, North Africa and Asia. Two species H. helix and H. nepalensis
are present in Pakistan. The H. nepalensis is locally known as Arbambal.19
2.1.2. Hedera nepalensis K. Koch
H. nepalensis is a species of the genus Hedera at altitude of about 1000-3000 m.
Plants grow up to 30 m in height, with simple leaves ranging from 2-15 cm long and yellow
flowers. Stem is creeping or climbing to a height of 30 m with adventitious roots. Flower
stalks (length 7-12 mm) and flowering hairy. Petals are yellow crown, stamens are 5 in
number, anthers are 1-2 mm long and pillar is short neck. Fruit is a drupe, flattened, 5-7 mm
Chapter 2 Plant introduction (Part-A)
8
long, 5-10 mm wide, with orange to red. The plant blooms from October to April. It occurs
mostly in moist soil in shade, at the height of 1000-3000 m and extensively climbs on walls,
rocks, tree trunks by its aerial roots used as a ground cover or decorative climber in gardens
and parks.20
2.1.3. Chemical constituents of the genus Hedera
In 1974 Mahran and his research group reported that the genus Hedera mainly consists
of α-hederin, hederasaponin B and hederasaponin C.21 In 1975 the same group isolated an
alkaloid named emetine from the methanolic extract of H. helix in Egypt.22 Mineo Shimizu
and his research group in 1978 isolated four saponins saponin K3, saponin K6, saponin K10
and saponin K12 from H. rhombea.23 Kizu, H. and his co-workers in 1985 isolated and
characterized four triterpene glycosides named Kizuta saponin K4, K5, K7 and K7C from the
stem bark of the Hedera rhombea.24 In 1985, Kizu, H. and his research group isolated
twelve saponins from the stem bark of H. nepalensis.25 Chandel, R. and Rostogi, R. in 1989,
isolated two saponins from H. nepalensis.26 In the same year Rank Gafner et.al., isolated a
diacetylene, 11 dehydrofaicarinol while Elias, R. and his co-workers in 1991 isolated four
triterpenoids saponins from the leaves of H. helix.27,28 In 1991 Lars, P. C. et.al., isolated
polyacetylenicepoxide from the fruits of H. helix.29 Kwon, B. and his research group in
1997 isolated a rhombenone; dammarane analogue containing 25-oxo from the leaves of H.
rhombea.30 In 2000 Bedir, E. and his research group isolated six triterpene saponins from the
fruits of H. helix.31 Mshvildadze, V. and his research group in 2001 isolated ten triterpenoid
saponins from the berries of H. colchica.32 Subsequently Mshvildadze, V. et.al., in 2005
reported arjunolic acid derivative glycoside from the stems of H. colchica.33 Yamazoe, S. in
Chapter 2 Plant introduction (Part-A)
9
2007 isolated two polyacetylenes from the extract of Japanese ivy (H. rhombea) flower
buds.34 Sieben, A. and his research group in 2009 isolated three saponins from the leaves
extract of ivy (H. helix).35 The literature survey of the genus Hedera regarding
phytochemistry is summarized in the Table: 2.1.
Table 2.1: Chemical constituents of the genus Hedera
S.
No.
Compounds Species References
1 α-Hederin H. helix 21
2 Hederasaponin B H. helix 21
3 Hederasaponin C H. helix 21
4 Emetine H. helix 22
5 Saponin K3 H. rhombea 23
6 Saponin K6 H. rhombea 23
7 Saponin K10 H. rhombea 23
8 Saponin K12 H. rhombea 23
9 Saponin K4 H. rhombea 24
10 Saponin K5 H. rhombea 24
11 Saponin K7 H. rhombea 24
12 Saponin K7c H. rhombea 24
13 Saponin A H. nepalensis 25
14 Saponin B H. nepalensis 25
15 Saponin D1 H. nepalensis 25
Chapter 2 Plant introduction (Part-A)
10
16 Saponin D2 H. nepalensis 25
17 Saponin E H. nepalensis 25
18 Saponin F H. nepalensis 25
19 Saponin H H. nepalensis 25
20 Saponin I H. nepalensis 25
21 Saponin K H. nepalensis 25
22 Saponin M H. nepalensis 25
23 Saponin N H. nepalensis 25
24 Saponin P H. nepalensis 25
25 Nepalin-1 H. nepalensis 26
26 Nepalin-2 H. nepalensis 26
27 Diacetylene, 11 dehydrofaicarinol H. helix 27
28 Hederasaponin E H. helix 28
29 Hederasaponin F H. helix 28
30 Hederasaponin H H. helix 28
31 Hederasaponin I H. helix 28
32 Polyacetylenicepoxide H. helix 29
33 Rhombenone H. rhombea 30
34 3-O-β-D-Glu-copyranosyl hederagenin H. helix 31
35 3-O-β-D-Glucopyranosyl-(1→2)-β-D-glucopyranosyl
oleanolic acid
H. helix 31
36 3-O-β-D-Glucopyranosyl-(1→2)-β-D-glucopyranosyl
hederagenin
H. helix 31
Chapter 2 Plant introduction (Part-A)
11
37 3-O-β-D-Glucopyranosyl hederagenin-28-O-β-D
-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester
H. helix 31
38 Helixosides A H. helix 31
39 Helixosides B H. helix 31
40 Saponin 1 H. colchica 32
41 Colchisides A H. colchica 32
42 Saponin 2 H. colchica 32
43 Saponin 4 H. colchica 32
44 Heteroside E2 H. colchica 32
45 Staunoside A H. colchica 32
46 Heteroside I H. colchica 32
47 Scheffleraside II H. colchica 32
48 Hederasaponin D H. colchica 32
49 Colchisides B H. colchica 32
50 Arjunolic acid derivative glycoside H. colchica 33
51 Polyacetylenes H. rhombea 34
52 (Z)-9,10-Epoxy-1-heptadecene-diyn-3-one H. helix 35
53 Falcarinone H. helix 35
54 Falcarinol H. helix 35
Chapter 2 Plant introduction (Part-A)
12
2.1.4 Structures of selected compounds reported from the genus
Hedera
α-Hederin
Hedersaponin B
Chapter 2 Plant introduction (Part-A)
13
Hedersaponin C
Hedersaponin D
Emetine
Chapter 2 Plant introduction (Part-A)
14
Saponin K3
OO
OH
O
CH2OH
COOH
OH
O
HO OH
O OH
Saponin K6
Chapter 2 Plant introduction (Part-A)
15
OO
OR
OR
OH
CH2OH
CO
O
OOO
CH2OR
OR
RO
OR
OR
OOOR
OR OR
Me
Saponin K10
Saponin K4
Chapter 2 Plant introduction (Part-A)
16
Saponin K5
Saponin K7
Saponin K7c
Chapter 2 Plant introduction (Part-A)
17
3-O-β-D-Glu-copyranosyl hederagenin
3-O-β-D-Glucopyranosyl-(1→2)-β-D-glucopyranosyl oleanolic acid
Chapter 2 Plant introduction (Part-A)
18
3-O-β-D-Glucopyranosyl-(1 → 2)-β-D-glucopyranosyl hederagenin
3-O-β-D-Glucopyranosyl hederagenin-28-O-β-D-glucopyranosyl-(1 → 6)-β-D-
glucopyranosyl ester
Helixosides A
Chapter 2 Plant introduction (Part-A)
19
Helixosides B
Colchiside A
Polyacetylene
Chapter 2 Plant introduction (Part-A)
20
2.1.5 Medicinal and pharmacological properties of the genus Hedera
The genus Hedera is known for its economical importance. Acute and chronic anti-
inflammatory profile of H. helix, in rats was reported by Suleyman and his group in 2003.36
The aqueous and methanolic extracts of H. helix were found to reduce blood glucose level in
rabbits to a significant level.37 The plant is traditionally used in folk medicine. It has been
reported that the leaves and berries of H. nepalensis are stimulating, diaphoretic, cathartic
and also used to treat indolent ulcers and abscesses.38 Qureshi and his research group also
reported that the decoction of the leaves is very effective against lice.39 Timen, D. et.al.,
reported the antifungal activity of H. helix extract.40 Agarwal and Rostogi investigated
antimitotic activity of glycosides isolated from the genus Hedera.41 Antileishmanial activity
of the saponins isolated from the H. helix were reported by Majester Savornin and his co-
workers.42 Cioaca et.al., reported the antibacterial activity of the saponins of the H. helix
which are more active against Gram positive bacteria as compared to Gram negative
bacteria.43 Quetin and his researcher investigated cytotoxicity both in vitro and in vivo on
Ehrlich tumor cells of the crude extract of H. helix.44 H. nepalensis leaves are traditionally
used for diabetes treatment.45 Hamayun et.al., in 2006 documented that the leaves of H.
nepalensis are used for the cancer treatment.46 Shah et.al., in 2006 reported that the plants
has hypoglycemic properties and is very effective against rheumatism, fever and pulmonary
infections.47 Inayatullah and his co-workers in 2007 screened the methanolic crude extract
of aerial part of the plant and tested for different bioassays such as cytotoxic, phytotoxic
activity, potato disc antitumor activity and brine shrimp.48 It has been considered one of the
most useful plant containing saponins for the treatment of cough and human ailments.49 The
literature survey showed that saponins from the leaves of H. helix have spasmolytic,
Chapter 2 Plant introduction (Part-A)
21
antifungal, anthelmintic, molluscicidal, antileishmanial and antimutagenic activities.50 The
fresh leaves and fruits of the H. helix are toxic and cause gastrointestinal irritation,
dermatitis and bloody diarrhea.51
Chapter 3
Results & Discussion
Chapter 3 Results & discussions, (Part A)
22
3.1. Present work
Due to medicinal and biological importance assigned to genus Hedera, chemical
explorations of H. nepalensis were carried out in current studies which resulted in the
isolation and structure determination of one new and four hitherto unreported compounds
from H. nepalensis. Structure of the new compound was identified by advanced
spectroscopic methods including UV, IR, MS, 1D (1H- and 13C-NMR; BB and DEPT) and
2D NMR (J- resolved, COSY-45o, NOESY, HSQC, HMBC) experiments. The known
compounds were determined by comparison their spectral data (1D and 2D NMR) with the
reported compounds in the literature.
3.2. New compound isolated from H. nepalensis
Hepalensiside A (1)
CH2OH
O
O
O
O
O
COOH
HO
HO
HOH
H3CHO
HOHO OH
O
Chapter 3 Results & discussions, (Part A)
23
3.3. Hitherto unreported compounds from H. nepalensis
Colchiside A (2)
Pastuchoside A (3)
Chapter 3 Results & discussions, (Part A)
24
Helixoside A (4)
Kizuta saponin K12 (5)
OCH2OH
OOH
HOO
O
HOHO
OH
O
O
O
O
OHO
OHHO
O
OHHO
OH
H3C
O
OHHO
OH
H3C
Chapter 3 Results & discussions, (Part A)
25
3.2.1. Hepalensiside A (1)
Compound 1 was obtained as a white needle crystal with melting point 235-236
°C. Its molecular formula was determined to be C46H72O16 from the pseudo molecular ion
peak at m/z 903.3012 (calcd. for C46H72O16 880.2313) in HRESI-MS together with the 1H-
and 13C-NMR data.
The 13C-NMR spectrum of compound 1 displayed 46 carbon signals of which 30 could
be attributed to aglycone moeity. Spectroscopic analysis including proton, 13C-NMR (Table:
4.1 vide experimental) and 2D NMR experiments showed that compound 1 is a sapogenin
triterpenoid. The 1H-NMR spectrum showed six tertiary methyl groups at δH 0.86 (3H, s, H-
30), 0.93 (3H, s, H-29), 0.99 (3H, s, H-25), 1.03 (3H, s, H-27), 1.07 (3H, s, H-26) and δH 1.09
(3H, s, H-24) and the corresponding 13C-NMR signals at δC 24.3 (C-30), 32.3 (C-29), 18.8 (C-
25), 20.1 (C-27), 17.2 (C-26), and δC 13.5 (C-24). Two olefinic protons were observed at δH
6.63 (1H, dd, J =10.5, 4.5 Hz, H-11), 5.73 (1H, d, J =10.5 Hz, H-12) while the olefinic
carbons were present at δC 125.9 (C-11), 127.1 (C-12), 133.2 (C-13), and δC 136.6 (C-18). A
pair of hydroxy methylene protons appeared at δH 3.91 and 4.32 connected to a carbon at δC
Chapter 3 Results & discussions, (Part A)
26
63.9 (C-23) in HSQC spectrum while a carboxylic carbon appeared at δC 178.9 (C-28) which
is a characteristic peak in hederagenin type of skeleton. The sugar moieties were identified as
L-arabinose, L-rhamnose and D-ribose in a ratio of 1:1:1 by acidic hydrolysis followed by GC
analysis and comparison the corresponding aldononitrile peracetates with the authentic
samples prepared in the manner described in the literature.52 The assignments of the NMR
signals associated with the aglycone moiety were made from HSQC, HMBC, 1H–1H COSY,
and NOESY experiments. The data revealed that the 1 has common hederagenin aglycone
skeleton. The two double bonds located at position 11 and position 13 was confirmed by the
HMBC spectrum (Figure: 3.1). A downfield shift of carbon C-3 was resonating at δC 81.2
indicated that oligosaccharide moiety attached at position C-3. The HMBC spectrum
confirmed the interglycosidic connectivities of 1 through correlations of anomeric proton at
δH 5.05 (H-1') of arabinose and C-3 (δC 81.2) of aglycon, the anomeric proton of rhamnose at
δH 6.32 (H-1'') with C-2' of arabinose (δC 75.3), similarly the anomeric proton of ribose at δH
5.96 (H-1''') showed connectivity with C-3'' (δC 81.2) of rhamnose. In view of the above
evidences the structure of 1 was established as 3β-O-[β-D-ribopyranosyl-(1→3)-α-L-
rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl] hederagenin-11,13-dien-28-oic acid.
Chapter 3 Results & discussions, (Part A)
27
Figure: 3.1 Important HMBC (H→C) correlations of compound 1
3.3.1. Colchiside A (2)
Compound 2 was obtained as white powder from the butanolic fraction. The HRESI-
MS indicated the molecular ion peak at m/z 604. 1171 a.m.u representing the molecular
formula C35H56O8 (calcd. for C35H56O8 604.1230).The NMR data (Table: 4.2 vide
experimental) showed that one anomeric carbon resonated at δC 106.3 and anomeric proton
resonated at δH 5.12. The carbon C-3 resonated at δC 83.4 and C-28 at δC 182.6 confirmed that
the sugar chain attached to C-3. The structure was elucidated as 3-O-(β -D-xylopyranosyl)-
Chapter 3 Results & discussions, (Part A)
28
hederagenin. The spectroscopic data (1H- and 13C-NMR) was in accordance with the data
reported for Colchiside A in the literature.53
3.3.2. Pastuchoside A (3)
Pastuchoside 3 was isolated as white powder. The molecular formula C53H86O21 was
determined on the basis HRESI-MS m/z 1528.7190 a.m.u (calcd. for C71H116O35 1528.7186).
The NMR data (Table: 4.3, 4.4 vide experimental) and acid hydrolysis of 3 yielded glucose,
arabinose and rhamnose as sugars and the skeleton was hederagenin. Therefore, the structure
of pastuchoside A (3) was determined as 3β -O-{α -L-rhamnopyranosyl-(1→2)-[α-
Lrhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)]-α-Larabinopyranosyl}-28-O-[α-L-
rhamnopyranosyl-(1→4)-β-Dglucopyranosyl-(1→6)-β-D-glucopyranosyl]-hederagenin. The
spectral data (1H- and 13C-NMR) was identical with the data reported for Pastuchoside A (3)
in the literature.54
Chapter 3 Results & discussions, (Part A)
29
3.3.3. Helixoside A (4)
Compound 4 was afforded from the butanolic fraction as a colourless amorphous
powder. The HRESI-MS indicated molecular ion peak at m/z 1143.0221 [M+Na]+
corresponding to the molecular formula C54H88O24 (calcd. for C54H88O24 1143.0217). The IR
spectrum for 4 showed an absorption bands for hydroxyl at 3410 cm-1, 1696 cm-1 for
carboxylic group and 1732 cm-1 for ester group. The proton and 13C-NMR data (Table: 4.5,
4.6 vide experimental) were in complete agreement with the data reported for Helixoside A
(4) in the literature and characterized as 3-O-β-D-glu-copyranosyl hederagenin.55
Chapter 3 Results & discussions, (Part A)
30
3.3.4. Kizuta saponin K12 (5)
OCH2OH
OOH
HOO
O
HOHO
OH
O
O
O
O
OHO
OHHO
O
OHHO
OH
H3C
O
OHHO
OH
H3C
Compound 5 was obtained as white powder from the butanolic fraction. The HRESI-MS
indicated molecular ion peak at m/z 1198.1593 a.m.u. (calcd. for C59H90O25 1198.1589)
showing the molecular formula C59H90O25. The IR spectrum for 5 showed an absorption
bands at 3414 cm-1 for hydroxyl, 1704 cm-1 for carboxylic group and 1739 cm-1 for ester
group. The proton and 13C-NMR data (Table: 4.7, 4.8 vide experimental) were in complete
agreement with the data reported for Kizuta saponin K12 (5) in the literature.56
3.4. Biological studies
3.4.1 Anti-bacterial assay
Various fractions n-hexane (G1), dichloromethane (H1), ethyl acetate (G2) and
methanol (H2) of stem and aerial parts of H. nepalensis were examined for anti-bacterial
assay against the four selected bacterial stains; Escherichia coli, Bacillus cereus, Proteus
mirabilis, Staphylococcus aureus, which showed good activity with zone of inhibition
Chapter 3 Results & discussions, (Part A)
31
ranging from 9-13 mm at concentration of 28 μg/ml (Table: 4.9 vide experimental). The G1
fractions showed good activity against P. mirabilis at conc. of 12 μg/ml while H2 and H3
exhibited good activity against B. cereus and E. coli at conc. of 13, 11 μg/ml respectively.
The remaining fraction (G-2, G3) was found almost inactive against tested pathogen.
3.4.2. Anti-fungal assay
Various fractions of the stem and aerial parts of H. nepalensis were also tested
against selected fungal strains (Candida albicans, Aspergillus flavus, Microsporum canis,
Fusarium solani and candida glabrata). None of the tested extract/ fractions showed
significant activity. (Table: 4.10 vide experimental).
3.4.3. Phytotoxicity assay
Different fractions of the stem and aerial parts of H. nepalensis were examined for
their in vitro phytotoxic bioassay. Two fractions of the aerial parts of H. nepalensis revealed
significant activities at highest doses against Lemna minor plant. Dichloromethane extract
(H1) of the stem of H. nepalensis revealed good activity, while MeOH and MeOH: H2O
(1:1) extracts revealed moderate activities at highest doses. (Table: 4.11 vide experimental).
3.4.4. Insecticidal assay
Insecticidal assay of the various fractions of the stem and aerial parts of H.
nepalensis were carried out by contact toxicity method.57 Neither of the fractions showed
significant activities (Table: 4.12 vide experimental).
Chapter 3 Results & discussions, (Part A)
32
3.4.5. Brine shrimp (Artemia salina) lethality bioassay
Various fractions of the stem and aerial parts of H. nepalensis were evaluated
for brine shrimp (Artemia salina) lethality bioassay. The n-hexane and EtOAc fractions
showed non-significant results, while the methanolic fraction was found to be cytotoxic at
highest dose (1000 μg/mL). Similarly dichloromethane fraction of the stem of H. nepalensis
showed no cytotoxicity. (Table: 4.13 vide experimental).
Chapter 4
Experimental
Chapter 4 Experimental (Part-A)
33
4.1. General experimental
Infrared spectra (IR) were obtained in solid state through FT/IR-4200 spectrometer.
Ultraviolet spectra (UV) were recorded on UNICAM UV 300 spectrophotometer. Optical
rotations were recorded on AA-100 polarimeter in chloroform at 20°C. Melting points of the
samples were recorded on Buchi 535 melting point apparatus. Finnigan MAT 112 11/34
computer system was used for recording of mass spectra. High resolution mass spectra
(HRMS) were employed for accurate mass measurement.
1H- and 13C-NMR spectra were recorded on Bruker AVANCE-400, AVANCE-600
MHz for 1H and 100, 125 MHz for 13C nuclei with TMS (tetramethylsilane) as internal
reference using CDCl3 as a solvent, the chemical shifts () values are given in ppm while J
values are given in Hz. Column chromatography was performed on silica gel (Si 60, 70-230
mesh) and precoated aluminum cards (0.2 mm thickness) with silica gel 60PF254 (Merck)
were used. Purity of the samples was checked by TLC and visualized by checking in UV
light and by spraying with I2 vapours and ceric sulphate solution.
4.2. Plant Material
The leaves of Hedera nepalensis were collected from Bara Gali, Khyber
Pakhtunkhwa, Pakistan in July 2009. The plant was identified by Abdul Majid of the
Department of Botany, Hazara University, Khyber Pakhtunkhwa, Pakistan. A voucher
specimen No. BT-576 was deposited in the herbarium of Hazara University.
Chapter 4 Experimental (Part-A)
34
4.3. Extraction and isolation
Leaves of H. nepalensis (400 g) were repeatedly (x3) extracted at room temperature
with methanol. After concentration under vacuum the syrupy liquid was treated with n-
BuOH to get a crude extract of saponins (80 g) which was subjected to column
chromatography on silica gel with solvent system CHCl3-MeOH-H2O ( 26:14:3) to afford
three fractions (1-3). Fraction 3 the most polar triterpene was further subjected to high
performance liquid chromatography (HPLC) eluted with MeOH-H2O (20% to 80% of
MeOH) to yield compound 1 (20 mg), compound 2 (26 mg), compound 3 (38 mg) and
mixture of 4 and 5 (85 mg), which was further subjected to column chromatography
afforded compound 4 (19 mg) and 5 (32 mg) (Scheme: 4.1).
Chapter 4 Experimental (Part-A)
35
Percolation with MeOH(three times) at room temp.
Not pursued
Leaves of H. nepalensis(400 g)
n-BuOH
Crude extract of saponins(80 g)
Column chromatography
Not pursued
HPLC(500 mg offraction 3)
1 (20 mg) 2 (26 mg) 3 (38 mg) mixture(85 mg)
Column chromatography
4 (19 mg) 5 (32 mg)
Fraction 2(35 g)
Fraction 3(12 g)
Fraction 1(32 g)
Aqueous layer (140 g)
Scheme: 4.1: Extraction and isolation from H. nepalensis K. Koch
Chapter 4 Experimental (Part-A)
36
4.4. Characterization of chemical constituents
Hepalensiside A (1)
Physical data
Yield: 20 mg
UVλmax ( in MeOH: 242 (4.62) nm
IR max cm-1: 1742, 1754 (ester/ lactone carbonyls), 1630 (C=C)
HR-ESI-MS: 903. 3012 [M+Na]+ (calcd. for C46H72O16 880.2313)
1H- and 13C-NMR : Table- 4.1.
Chapter 4 Experimental (Part-A)
37
Table: 4.1. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 1 in C5D5N
Position δH Multiplicity (J in Hz) δC Multiplicity
1a /1b 1.16/1.89 m 38.6 CH2
2a/2b 2.05/2.30 m 25.8 CH2
3 4.28 dd (11.5, 4.2) 81.2 CH
4 - - 42.8 C
5 1.75 m 47.6 CH
6a /6b 1.43/1.72 m 18.4 CH2
7a/7b 1.25/1.39 m 32.7 CH2
8 - - 42.7 C
9 2.11 m 53.8 CH
10 - - 36.7 C
11 6.63 dd (10.5, 4.5) 125.9 CH
12 5.73 d (10.5) 127.1 CH
13 - - 133.2 C
14 - - 42.2 C
15a /15b 1.02/1.93 m 25.7 CH2
16a / 16b 1.78/2.20 m 33.5 CH2
17 - - 48.9 C
18 - - 136.6 C
19a / 19b 2.11/2.68 m 41.2 CH2
20 - - 32.9 C
21a/21b 1.32/1.68 m 37.8 CH2
Chapter 4 Experimental (Part-A)
38
22a / 22b 1.48/2.58 m 36.4 CH2
23a/23b 3.91/4.32 m 63.9 CH2
24 1.09 s 13.5 CH3
25 0.99 s 18.8 CH3
26 1.07 s 17.2 CH3
27 1.03 s 20.1 CH3
28 - - 178.9 C
29 0.93 s 32.3 CH3
30 0.86 s 24.3 CH3
1' 5.05 d (6.8) 104.8 CH
2' 4.01 dd (10.2, 6.8) 75.3 CH
3' 4.58 m 75.3 CH
4' 4.11 m 69.7 CH
5'a/5'b 3.65/4.23 d (10.8)/ m 47.6 CH2
1'' 6.32 d (6.2) 101.3 CH
2'' 4.92 dd (10.7, 6.2) 72.1 CH
3'' 4.76 m 81.2 CH
4'' 4.35 m 72.4 CH
5'' 4.17 m 70.3 CH
6'' 1.53 d (6.4) 18.3 CH3
1''' 5.96 d (6.6) 104.6 CH
2''' 4.41 dd (10.3, 6.6) 72.7 CH
Chapter 4 Experimental (Part-A)
39
3''' 4.47 m 68.3 CH
4''' 4.10 m 69.7 CH
5a'''/5'''b 4.12/4.32 dd (10.5, 3.2)/m 65.2 CH2
Colchiside A (2)
Physical data
Yield: 26 mg
UVλmax ( in MeOH: 245 (4.87) nm
IR max cm-1: 3401 (hydroxyl), 3056 (C-H stretching
aromatic), 2917 (C-H stretching aliphatic)
HR-ESI-MS: 604.1171 (calcd. for C35H56O8 604.1230)
1H- and 13C-NMR : Table- 4.2.
Chapter 4 Experimental (Part-A)
40
Table: 4.2. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 2 in C5D5N
Position δH Multiplicity (J in Hz) δC Multiplicity
1a /1b 1.16/1.89 m 38.6 CH2
2a/2b 2.05/2.30 m 25.8 CH2
3 4.28 dd (10.8, 4.1) 83.4 CH
4 - - 42.8 C
5 1.75 m 47.6 CH
6a /6b 1.43/1.72 m 18.4 CH2
7a/7b 1.25/1.39 m 32.7 CH2
8 - - 42.7 C
9 2.11 m 53.8 CH
10 - - 36.7 C
11a/11b 1.92 dd (10.5, 3.3) 125.6 CH2
12 5.68 d (10.5) 127.3 CH
13 - - 134.1 C
14 - - 42.2 C
15a /15b 1.02/1.93 m 25.7 CH2
16a / 16b 1.78/2.20 m 33.5 CH2
17 - - 48.9 C
18 1.48 m 45.7 CH
19a / 19b 2.11/2.68 m 41.2 CH2
20 - - 32.9 C
21a/21b 1.32/1.68 m 37.8 CH2
Chapter 4 Experimental (Part-A)
41
22a / 22b 1.48/2.58 m 36.4 CH2
23a/23b 3.90/4.32 m 62.3 CH2
24 1.09 s 13.5 CH3
25 0.95 s 18.4 CH3
26 1.04 s 16.7 CH3
27 0.97 s 19.8 CH3
28 - - 182.6 C
29 0.93 s 30.5 CH3
30 0.86 s 26.7 CH3
1' 5.12 d (6.8) 106.3 CH
2' 4.03 dd (10.7, 6.8) 75.4 CH
3' 4.38 m 78.2 CH
4' 4.21 m 71.7 CH
5'a/5'b 3.45/4.31 dd (10.5, 2.2)/ m 66.8 CH2
Chapter 4 Experimental (Part-A)
42
Pastuchoside A (3)
Physical data
Yield: 38 mg
UVλmax (in MeOH: 259 (4.56) nm
IR max cm-1: 3432 (hydroxyl), 3055 (C-H stretching
aromatic), 2920 (C-H stretching aliphatic)
HR-ESI-MS: 1528.7190 a.m.u (calcd. for C71H116O35 1528.7186)
1H- and 13C-NMR : Table- 4.3, 4.4.
Chapter 4 Experimental (Part-A)
43
Table: 4.3. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 3 in C5D5N
Position δH Multiplicity (J in Hz) δC Multiplicity
1a /1b 1.06/1.75 m 39.6 CH2
2a/2b 2.04/2.28 m 26.8 CH2
3 4.14 dd (10.6, 4.4) 81.7 CH
4 - - 43.9 C
5 1.73 m 48.7 CH
6a /6b 1.46/1.62 m 18.3 CH2
7a/7b 1.28/1.32 m 33.5 CH2
8 - - 40.7 C
9 2.13 m 49.8 CH
10 - - 36.9 C
11a/11b 1.88 dd (10.3, 2.4) 24.6 CH2
12 5.63 d (10.3) 127.3 CH
13 - - 142.5 C
14 - - 42.7 C
15a /15b 1.04/1.91 m 27.8 CH2
16a / 16b 1.73/2.18 m 24.2 CH2
17 - - 48.4 C
18 1.49 m 42.3 CH
19a / 19b 2.13/2.62 m 47.2 CH2
20 - - 31.7 C
21a/21b 1.33/1.59 m 34.4 CH2
Chapter 4 Experimental (Part-A)
44
22a / 22b 1.42/2.53 m 33.2 CH2
23a/23b 3.43/4.36 m 64.3 CH2
24 1.05 s 13.3 CH3
25 0.93 s 17.4 CH3
26 1.03 s 17.7 CH3
27 0.95 s 20.7 CH3
28 - - 178.2 C
29 0.92 s 32.8 CH3
30 0.88 s 23.3 CH3
1' 4.48 d (6.6) 106.32 CH
2' 3.73 dd (11.3, 6.6) 75.4 CH
3' 3.84 m 78.2 CH
4' 4.02 m 71.7 CH
5'a/5'b 4.15/3.54 dd (10.6, 2.7)/ m 66.8 CH2
1'' 5.36 d (6.4) 102.7 CH
2'' 3.97 dd (10.4, 6.4) 72.6 CH
3'' 3.84 m 72.5 CH
4'' 4.35 dd (6.8, 3.1) 73.9 CH
5'' 3.53 m 70.6 CH
6'' 1.45 d (6.4) 18.2 CH3
1''' 6.22 d (6.7) 105.5 CH
2''' 4.94 dd (10.1, 6.7) 75.3 CH
Chapter 4 Experimental (Part-A)
45
3''' 5.45 m 76.4 CH
4''' 3.94 m 79.4 CH
5''' 3.87 m 76.6 CH
6'''a/6'''b 4.43/4.26 dd (12.3, 2.8)/m 61.8 CH2
1'''' 4.75 d (6.7) 101.8 CH
2'''' 3.75 m 72.9 CH
3'''' 3.85 m 72.3 CH
4'''' 3.41 dd (7.3, 1.4) 73.3 CH
5'''' 4.04 m 70.8 CH
6'''' 1.32 d (6.7) 17.3 CH3
Table: 4.4. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 3 for sugar moieties
at position C-28
1'a/1'b 5.36 dd (6.9, 2.6)/m 95.8 CH2
2' 3.33 m 73.6 CH
3' 3.55 m 78.2 CH
4' 3.46 m 70.8 CH
5' 3.62 m 78.3 CH
6'a/6'b 4.12/3.65 dd (11.5, 2.8)/m 69.4 CH2
1'' 4.43 d (7.3) 104.5 CH
2'' 3.26 dd (10.4, 7.3) 75.6 CH
3'' 3.45 m 76.4 CH
4'' 3.56 m 79.4 CH
Chapter 4 Experimental (Part-A)
46
5'' 3.37 m 76.4 CH
6''a/6''b 3.45/3.75 dd (11.2, 2.7)/m 61.6 CH2
1''' 4.62 d (6.8) 102.4 CH
2''' 3.64 m 72.7 CH
3''' 3.87 dd (7.6, 3.4) 73.3 CH
4''' 3.47 m 73.6 CH
5''' 4.07 m 70.9 CH
6''' 1.28 d (6.4) 18.5 CH3
Chapter 4 Experimental (Part-A)
47
Helixoside A (4)
Physical data
Yield: 19 mg
UVλmax (in MeOH: 272 (4.72) nm
IR max cm-1: 3442 (hydroxyl), 3035 (C-H stretching
aromatic), 2932 (C-H stretching aliphatic)
HR-EI-MS: 1143.0221 [M+Na]+ (calcd. for C54H88O24 1120.0132)
1H- and 13C-NMR : Table- 4.5, 4.6.
Chapter 4 Experimental (Part-A)
48
Table: 4.5. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 4 in C5D5N
Position δH Multiplicity (J in Hz) δC Multiplicity
1a /1b 1.04/1.74 m 39.7 CH2
2a/2b 2.02/2.26 m 26.3 CH2
3 3.84 dd (10.2, 4.2) 84.7 CH
4 - - 43.5 C
5 1.71 m 48.8 CH
6a /6b 1.44/1.64 m 18.4 CH2
7a/7b 1.26/1.33 m 33.6 CH2
8 - - 40.5 C
9 2.14 m 49.4 CH
10 - - 36.5 C
11a/11b 1.94 dd (10.4, 2.4) 24.4 CH2
12 5.64 d (10.4) 126.3 CH
13 - - 143.5 C
14 - - 42.5 C
15a /15b 1.02/1.92 m 28.3 CH2
16a / 16b 1.74/2.16 m 24.6 CH2
17 - - 48.5 C
18 1.53 m 42.3 CH
19a / 19b 2.12/2.60 m 47.4 CH2
20 - - 31.3 C
21a/21b 1.32/1.58 m 34.3 CH2
Chapter 4 Experimental (Part-A)
49
22a / 22b 1.40/2.54 m 33.4 CH2
23a/23b 3.46/4.35 m 64.5 CH2
24 1.08 s 13.4 CH3
25 0.97 s 17.4 CH3
26 1.01 s 17.7 CH3
27 0.93 s 20.8 CH3
28 - - 178.3 C
29 0.96 s 32.5 CH3
30 0.81 s 23.4 CH3
1' 4.52 d (7.5) 104.4 CH
2' 3.35 dd (10.4, 7.5) 81.5 CH
3' 3.46 m 77.6 CH
4' 3.57 m 71.3 CH
5' 3.58 m 78.4 CH
6'a/6'b 4.21/3.43 dd (11.2, 2.5)/m 62.4 CH2
1'' 4.69 d (7.4) 104.2 CH
2'' 3.34 dd (10.8, 7.4) 76.1 CH
3'' 3.67 m 77.8 CH
4'' 3.57 m 71.5 CH
5'' 3.35 m 78.5 CH
6''a/6''b 3.44/3.78 dd (11.6, 2.5) 62.9 CH2
Chapter 4 Experimental (Part-A)
50
Table: 4.6. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 4 for sugar moieties at
position C-28
1' 5.36 d (7.9) 95.7 CH
2' 3.26 dd (10.5, 7.9) 73.8 CH
3' 3.74 m 78.0 CH
4' 3.45 m 70.9 CH
5' 3.45 m 77.5 CH
6'a/6'b 4.02/3.34 dd (10.5, 2.4)/ m 69.5 CH2
1'' 4.35 d (7.8) 104.6 CH
2'' 3.32 dd (10.9, 7.8) 75.1 CH
3'' 3.64 m 77.6 CH
4'' 3.55 m 71.6 CH
5'' 3.25 m 78.0 CH
6''a/6''b 3.37/3.64 dd (11.3, 4.8)/m 62.7 CH2
Chapter 4 Experimental (Part-A)
51
Kizuta saponin K12 (5)
OCH2OH
OOH
HOO
O
HOHO
OH
O
O
O
O
OHO
OHHO
O
OHHO
OH
H3C
O
OH
HOOH
H3C
Physical data
Yield: 32 mg
UVλmax ( in MeOH: 254 (4.57) nm
IR max cm-1: 3414 (hydroxyl), 1704 for carboxylic group
1739 cm-1 for ester group 3035 (C-H stretching
aromatic), 2932 (C-H stretching aliphatic)
HR-ESI-MS: 1198.1593(calcd. for C59H90O25 1198.1589)
1H- and 13C-NMR : Table- 4.7, 4.8.
Chapter 4 Experimental (Part-A)
52
Table: 4.7. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 5 in C5D5N
Position δH Multiplicity (J in Hz) δC Multiplicity
1a /1b 1.03/1.72 m 38.5 CH2
2a/2b 2.01/2.20 m 25.7 CH2
3 3.86 dd (10.8, 4.3) 83.8 CH
4 - - 44.5 C
5 1.73 m 47.3 CH
6a /6b 1.35/1.54 m 18.6 CH2
7a/7b 1.30/1.32 m 33.1 CH2
8 - - 39.4 C
9 2.02 m 48.5 CH
10 - - 37.4 C
11 1.74 dd (10.2, 2.6)/ m 24.2 CH2
12 5.32 d (10.2) 124.4 CH
13 - - 142.7 C
14 - - 42.4 C
15a /15b 1.01/1.94 m 28.4 CH2
16a / 16b 1.72/2.13 m 24.5 CH2
17 - - 48.3 C
18 1.49 m 42.2 CH
19a / 19b 2.31/2.46 m 47.1 CH2
20 - - 31.2 C
21a/21b 1.31/1.53 m 34.2 CH2
Chapter 4 Experimental (Part-A)
53
22a / 22b 1.42/2.52 m 33.5 CH2
23a/23b 3.34/4.32 m 64.2 CH2
24 1.02 s 13.5 CH3
25 0.95 s 17.5 CH3
26 1.05 s 17.1 CH3
27 0.94 s 20.4 CH3
28 - - 178.1 C
29 0.92 s 32.2 CH3
30 0.78 s 23.3 CH3
1' 5.43 d (7.7) 102.5 CH
2' 3.65 m 73.3 CH
3' 3.79 m 71.4 CH
4' 4.53 m 73.8 CH
5' 3.46 m 70.2 CH2
1'' 4.52 d (7.4) 106.2 CH
2'' 3.74 dd (8.8, 7.4) 75.1 CH
3'' 3.87 m 78.1 CH
4'' 4.12 m 71.3 CH
5''a/5''b 4.21/3.45 d (10.4)/ m 66.4 CH
6'' 1.34 d (6.3) 18.5 CH3
Chapter 4 Experimental (Part-A)
54
Table: 4.8. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 5 for sugar moieties at
position C-28
1' 4.57 d (7.3) 102.3 CH
2' 3.51 dd (9.2, 7.3) 72.1 CH
3' 3.73 m 73.3 CH
4' 3.35 m 73.7 CH
5' 4.11 m 69.4 CH
6'a/6'b 1.19 dd (6.3, 2.1) /m 17.4 CH2
1'' 5.21 d (6.7) 96.1 CH
2'' 3.21 dd (10.5, 6.7) 72.9 CH
3'' 3.42 m 77.3 CH
4'' 3.51 m 70.2 CH
5'' 3.57 m 77.4 CH
6''a/6''b 4.09/3.49 dd (11.2, 2.4)/m 69.2 CH2
1''' 4.34 d (6.6) 104.1 CH
2''' 3.16 dd ( 10.3, 6.6) 75.3 CH
3''' 3.23 m 75.6 CH
4''' 3.34 m 79.2 CH
5''' 3.45 m 76.3 CH
6''' 1.26 d (7.2) 18.3 CH3
Chapter 4 Experimental (Part-A)
55
Table: 4.9. Anti-bacterial activity of H. nepalensis
Keywords: X = Non significant, aerial parts = G, stem = H, G1 = n-hexane, G2
EtOAc, G3 = MeOH, H1 = Dichloromethane, H2 = Methanol, H3 = MeOH: H2O
(1:1), P. m = Proteus mirabilis, S. a = Staphylococcus aureus, E. c = Escherichia
coli, B. c = Bacillus cereus
Fractions E. c B. s P. m S. a
G1 x x 12 X
G2 x x X X
G3 x x X X
H1 10 x X 9
H2 x 13 X X
H3 11 x X X
DMSO (–) x x X X
Imipenum 10 µg/Disc (+) 34 32 28 23
Chapter 4 Experimental (Part-A)
56
Table: 4.10. Anti-fungal profile of H. nepalensis
Fractions Name of Fungus % inhibition Std. Drug Mic (µg/mL)
G1
C. a 0 Miconazole (110.8)
A. f 0 Amphotericin B (20.20)
M. c 0 Miconazole (98.4)
F. s 13 Miconazole (73.25)
C. g 18 Miconazole (110.8)
G2
C. a 16 Miconazole (110.8)
A. f 0 Amphotericin B (20.20)
M. c 0 Miconazole (98.4)
F. s 21 Miconazole (73.25)
C. g 14 Miconazole (110.8)
G3
C. a 12 Miconazole (110.8)
A. f 14 Amphotericin B (20.20)
M. c 0 Miconazole (98.4)
F. s 0 Miconazole (73.25)
C. g 14 Miconazole (110.8)
H1
C. a 12 Miconazole (110.8)
A. f 0 Amphotericin B (20.20)
M. c 21 Miconazole (98.4)
F. s 16 Miconazole (73.25)
C. g 0 Miconazole (110.8)
H2 C. a 0 Miconazole (110.8)
Chapter 4 Experimental (Part-A)
57
Keywords: Aerial parts = G, stem = H, G1 = n-hexane, G2 = EtOAc, G3 = MeOH,
H1 = dichloromethane, H2 = Methanol, H3 = MeOH: H2O (1:1), C. a = Candida albicans,
A. f = Aspergillus flavus, M. c =Microsporum canis, F. s = Fusarium solani, C. g= Candida
glabrata
.
Table: 4.11. Phytotoxicity assay of H. nepalensis
Fractions
Compounds
Conc.
(µg/mL)
No. of fronds% Growth
regulation
Std. Drug
Conc.
(µg/mL)Samples Control
G1
1000 02
20
70
0.015100 01 30
10 0 10
G2 1000 09 70
A. f 15 Amphotericin B (20.20)
M. c 24 Miconazole (98.4)
F. s 19 Miconazole (73.25)
C. g 22 Miconazole (110.8)
H3
C. a 13 Miconazole (110.8)
A. f 0 Amphotericin B (20.20)
M. c 0 Miconazole (98.4)
F. s 21 Miconazole (73.25)
C. g 12 Miconazole (110.8)
Chapter 4 Experimental (Part-A)
58
100 14 20 20 0.015
10 17 10
G3
1000 07
20
70
0.015100 15 30
10 16 10
H1
1000 09
20
50
0.015100 11 40
10 16 15
H2
1000 10
20
45
0.015100 15 20
10 16 10
H3
1000 13
20
40
0.015100 15 20
10 16 10
. Keywords: Aerial parts = G, stem = H, G1 = n-hexane, G2 = EtOAc,
G3 = MeOH, H1 = dichloromethane, H2 = Methanol, H3 = MeOH: H2O (1:1)
Chapter 4 Experimental (Part-A)
59
Table: 4.12. Insecticidal assay of H. nepalensis
Fractions Name of Insects % Mortality
G1
Tribolium castaneum 0
Rhyzopertha dominica 0
Callosbruchus analis 20
G2
Tribolium castaneum 0
Rhyzopertha dominica 0
Callosbruchus analis 0
G3
Tribolium castaneum 0
Rhyzopertha dominica 0
Callosbruchus analis 0
H1
Tribolium castaneum 0
Rhyzopertha dominica 20
Callosbruchus analis 0
H2
Tribolium castaneum 0
Rhyzopertha dominica 0
Callosbruchus analis 0
H3
Tribolium castaneum 0
Rhyzopertha dominica 0
Callosbruchus analis 20
Keywords: Aerial parts = G, stem = H, G1 = n-hexane, G2 = EtOAc, G3 = MeOH,
H1 = dichloromethane, H2 = Methanol, H3 = MeOH: H2O (1:1)
Chapter 4 Experimental (Part-A)
60
Table: 4.13. Brine Shrimp lethality bioassay of H. nepalensis
FractionsDoses
(µg/mL)
No. of
shrimps
No. of
survivors
LD50
(µg/mL)STD. Drug
LD50
(µg/mL)
G1
1000 30 22
413892.80 Etoposide 7.4625100 30 25
10 30 26
G2
1000 30 25
4251653 Etoposide 7.4625100 30 27
10 30 28
G3
1000 30 16
1050.50 Etoposide 7.4625100 30 21
10 30 28
H1
1000 29 19
1039111.1 Etoposide 7.4625100 29 22
10 29 23
H2
1000 29 15
981.641 Etoposide 7.4625100 29 22
10 29 20
H3
1000 29 13
607.64 Etoposide 7.4625100 29 22
10 29 28
Keywords: Aerial parts = G, stem = H, G1 = n-hexane, G2 = EtOAc, G3 = MeOH,
H1 = dichloromethane, H2 = Methanol, H3 = MeOH: H2O (1:1)
Chapter 5
Plant Introduction
Cornus macrophylla Wall. Ex. Roxb
Chapter 5 Plant introduction (Part-B)
61
5.1. Plant Introduction
Plant description
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida
Order: Cornales
Family: Cornaceae
Genus: Cornus
Specie: C. macrophylla
Binomial name: Wall
5.1.1. Genus Cornus
Genus Cornus belongs to the family Cornaceae consists of 55 species, distributed in
the temperate region. It is represented in Pakistan by three species namely C. macrophylla,
C. capitata and C. oblonga. Cornus species are represented by shining colorful flowers and
fruits, generally grown as ornamental plants.58
5.1.2. Cornus macrophylla Wall
C. macrophylla is a medium sized tree, 12-15 m tall. Leaves are 7-15 cm long, 3- 9
cm broad, hairs are modified, apex acute 1.5- 4 cm long petioles. Stamens are equal or
slightly longer than petals, anthers are yellow or rarely blue. Cylindrical style slightly
expanded at apex to apparently clavate, 2-4 mm stigma subcapitate, broader than style
slightly lobed. Fruits are purplish black or bluish black. The flowers are hermaphrodite,
pollinated by insects. It has flowering period from April to June.
Chapter 5 Plant introduction (Part-B)
62
Suitable for light (sandy), medium (loamy) heavy (clay) soils and can be grown in
heavy clay soils. It can grow in semi-shade or no shade. It prefers moist soils. It is
distributed in Afghanistan, Japan, China, Himalayas Pakistan and India at 1500 m to 2700 m
elevation. It is a comparatively common in Pakistan.59
5.1.3. Chemical constituents of the genus Cornus
The earliest evidence regarding phytochemical studies on the genus Cornus trace
back to 1960 when Rudolf and Nair carried out chemical examination of C. Stolonifera, and
reported hyperin from its stem bark.60 In 1973, Du and Francis identified and characterized
anthocyanins, cyanidin 3-rhamnosylgalactoside, pelargonidin 3-galactoside, cyanidin 3-
galactoside, pelargonidin 3-rhamnosylgalactoside and delphinidin 3-galactoside by
chromatographic spectrophotometric and chemical analysis.61 In the same year Jensen and
his research group isolated cornin and dihydrocornin from the leaves of C. nuttallii.62
Chikaon Ishino and his co-workers in 1988 isolated a cytotoxic compound halleridone from
the fruits of C. controvers.63 Hatano, et.al., in 1989 isolated cornusiin A, cornusiin B,
cornusiin C, tellimagranin I, tellimagranin II, isoterchebin, gemin D, 1,2,3-tri-O-galloyl-β-
D-glucose, 1,2,6,-tri-O-galloyl-β-D-glucose, 2,3,-di-O-galloyl-D-glucose, 1,2,3,6-tetra-O-
galloyl-β-D-glucose and camptothin B from the fruits of C. officinalis, and characterized
them by chemical and spectroscopic analysis.64 In 1990 Takuo, et.al., also isolated
digalloylglucose from the fruits of C. officinalis.65 Hatano, et.al., in 1990 isolated further
compounds cornoside and cornusiin G and 7-O-galloylsecologanol from the fruits of C.
officinalis.66 In 1998, Slimestad and Andersen isolated four anthocyanins from the fruits of
C. suecica, and characterized their structure as cyanidin 3-O- chromatographic β-(2'-
glucopyranosyl chemical-O-β-galactopyranoside, cyanidin 3-O-β-(2'-glucopyranosyl-O-β-
Chapter 5 Plant introduction (Part-B)
63
gluco-pyranoside), cyanidin 3-glucoside and cyanidin 3-galactoside.67 Subsequently Kim
and Kwak isolated dimethyltetrahydrofuran cis-2, 5-dicarboxylate, a furan derivative from
the fruits of C. officinalis.68 In 1998, Stermitz and Krull studied iridoid glycosides;
scandoside methyl ester, Scandoside geniposide, monotropein, hastatoside and galioside
from the fruit of C. Canadensi.69 Lee, D. and his research group in 2000 reported phenolic
compounds; (-)-2,3-digalloyl-4-(E)-caffeoyl-L-threonic acid, (-)-2-galloyl-4-(E)-caffeoyl-L-
threonic acid, (-)-4-(E)-cafeoyl-L-threonic acid and kaempferol 3-O-α-L-rhamnoside from
the leaves of C. Controversa.70 Tanaka, et.al., in 2001 isolated iridoid glucosides; 6α-
dihydrocornic acid, 6β-dihydrocornic acid and 3, 3ꞌ-di-O-methylellagic acid 4-(5ꞌꞌ-acetyl)-α-
l-arabinofuranoside along with three similar compounds; stenophyllin H1, dihydrocornin
and cornin from the C. capitata adventitious roots culture.71 Tanaka and his research group
in 2003 isolated 1, 2, 3, 4, 6-penta-O-galloyl-β-D-glucose from the root of C. capitata.72
Vareed, et al., in 2006 isolated delphinidin 3-O-rutinoside, cyanidin 3-O- glucoside and
delphinidin 3-O-glucoside from the C. alternifolia.73 In 2007, Lee, D. and his research group
isolated flavonoids from the fruits of C. kousa as kaempferol, astragalin and isoquercitin.74
In 2008 Tural and Koca characterized major anthocyanins, cyanidin 3-O-rutinoside and
pelargonidin 3-O-glucoside from the fruits C. mas.75 In 2008 Lee, D. and his group isolated
and characterized a lignan glycoside, (7'S,8'R)-dihydrodehydroconiferyl alcohol-4'-O-β-D-
xylopyranoside along with its aglycone, (7'S,8'R)-dihydrodehydroconiferyl alcohol from C.
kausa.76 Pawlowska et.al., phytochemically studied the methanolic fruits extract of C. mas
and reported flavonoids, quercetin 3-O-xyloside, quercetin 3-O-rhamnoside, quercetin 3-O-
rutinoside, quercetin 3-O-galactoside, quercetin 3-O-glucoside, quercetin 3-O-glucuronide,
a kaempferol 3-O-galactoside and aromadendrin 7-O-glucoside.77 Subsequently Lee, D.
Chapter 5 Plant introduction (Part-B)
64
et.al., isolated cytotoxic triterpenoids from the fruits of C. Kausa and characterized as
arjunolic acid, asiatic acid, betulinic acid, betulinic aldehyde, lupeol, taraxasterol,
tormentic acid, ursolic acid, ursolic aldehyde, and 19-hydroxyasiatic acid.78 Recently Wali
Ullah isolated 3ꞌ-O-methyl-3,4-methylenedioxy ellagic acid, betulin, betulinic acid and
cornallegic acid from the stem bark of C. macrophylla.79 The compounds reported in the
literature from the genus Cornus are summarized in Table: 5.1.
Table: 5.1. Chemical constituents of the Genus Cornus
S. No. Compounds Species References
1 Hyperin C. stolonifera 61
2 Delphinidin 3-galactoside C. mas 62
3 Cyanidin 3-galactoside C. mas 62
4 Cyanidin 3-rhamnosylgalactoside C. mas 62
5 Pelargonidin 3-galactoside C. mas 62
6 Pelargonidin 3-rhamnosyl–galactoside C. mas 62
7 Dihydrocornin C. nuttallii 63
8 Cornin C. nattullii 63
9 Halleridone C. controvers 64
10 Cornusiin A C. officinalis 65
11 Cornusiin B C. officinalis 65
12 Cornusiin C C. officinalis 65
13 Tellimagranin I C. officinalis 65
14 Tellimagranin II C. officinalis 65
15 Isoterchebin C. officinalis 65
16 Gemin D C. officinalis 65
17 Camptothin B C. officinalis 65
18 1,2,3-Tri-O-galloyl-β-D-glucose C. officinalis 65
Chapter 5 Plant introduction (Part-B)
65
19 1,2,6-Tri-O-galloyl-β-D-glucose C. officinalis 65
20 1,2,3,6 –Tetra-O-galloyl-β-D-glucose C. officinalis 65
21 2,3,-di-O-galloyl-D-glucose C. officinalis 65
22 Digalloylglucose C. officinalis 66
23 Cornoside C. officinalis 67
24 Cornusiin G C. officinalis 67
25 7-O-galloylsecologanol C. officinalis 67
26 Cyanidin 3-O- chromatographic β-(2ꞌ-
glucopyranosyl, chemical –O-β-
galactopyranoside
C. suecica 68
27 Cyanidin 3-O-β-(2ꞌ -glucopyranosyl-O-β-
gluco-pyranoside)
C. suecica 68
28 Cyanidin 3-glucoside C. suecica, 68
29 Cyanidin 3-galactoside C. suecica 68
30 Dimethyltetrahydrofurancis-2,5dicarboxy-
late
C. officinalis 69
31 Monotropein C. canadensis 70
32 Hastatoside C. canadensis 70
33 Scandoside geniposide C. Canadensis 70
34 Galioside C. Canadensis 70
35 Scandoside methyl ester C. Canadensis 70
37 (-)-2,3-Digalloyl-4-(E)-caffeoyl-L-threonic
acid
C. controversa 71
38 (-)-2-Galloyl-4-(E)-caffeoyl-L-threonic acid C. controversa 71
39 (-)-4-(E)-Cafeoyl-L-threonic acid C. controversa 71
40 kaempferol 3-O-α-L-rhamnoside C. controversa 71
41 3,3'-Di-O-methylellagic acid 4-(5''-acetyl)-
α- L-arabinofuranoside
C. capitata 72
42 6α-dihydrocornic acid C. capitata 72
Chapter 5 Plant introduction (Part-B)
66
43 6β-dihydrocornic acid C. capitata 72
44 Stenophyllin H1 C. capitata 72
48 1,2,3,4,6-Penta-O-galloyl-β-D-glucose C. capitata 73
49 delphinidin 3-O-rutinoside C. alternifolia 74
50 delphinidin 3-O-glucoside C. alternifolia 74
51 cyanidin 3-O-glucoside. C. alternifolia 74
52 Kaempferol C. kousa 75
53 Astragalin C. kousa 75
54 Isoquercitin C. kousa 75
55 cyanidin 3-O-rutinoside C. mas 76
56 pelargonidin 3-O-glucoside C. mas 76
57 7'S,8'R-dihydrodehydroconiferyl alcohol-4'-
O-β-D-xylopyranoside
C. kousa 77
58 7'S,8'R-dihydrodehydroconiferyl alcohol C. kousa 77
59 Quercetin 3-O-xyloside C. mas 78
60 Quercetin 3-O-rhamnoside C. mas 78
61 Quercetin 3-O-rutinoside C. mas 78
62 Quercetin 3-O-galactoside C. mas 78
63 Quercetin 3-O-glucoside C. mas 78
64 Quercetin 3-O-glucuronide C. mas 78
65 Kaempferol 3-O-galactoside C. mas 78
66 Aromadendrin 7-O-glucoside C. mas 78
67 Lupeol C. kousa 79
68 Taraxasterol C. kousa 79
69 Betulinic acid C. kousa 79
70 Betulinic aldehyde C. kousa 79
71 Ursolic aldehyde C. kousa 79
72 Arjunolic acid C. kousa 79
73 Tormentic acid C. kousa 79
74 Asiatic acid C. kousa 79
Chapter 5 Plant introduction (Part-B)
67
75 19-Hydroxyasiatic acid C. kousa 79
76 3'-O-Methyl-3,4-methylenedioxy ellagic
acid
C. macrophylla 80
77 Betulin C. macrophylla 80
78 Cornallegic acid C. macrophylla 80
5.1.4. Structures of selected compounds reported from the genus
Cornus
Hyperin
Delphinidin 3-O-galactoside
Chapter 5 Plant introduction (Part-B)
68
O
OGlc
OCH3OOH
Cornin
1, 2, 3-Tri-O-galloyl-β-D-glucose
OH
O
O
H
OHOHH
H
O O
HO
HOOH
HO
O
HO
HOHO
HOHO
HO
1, 2, 6-Tri-O-galloyl-β-D-glucose
Chapter 5 Plant introduction (Part-B)
69
OH
HO
O
H
OOH
H
O O
O
HO
HOOH
OH OH
OH
O
HOOH
OH
HO
HO
HOHO
1, 2, 3, 6-Tetra-O-galloyl-β-D-glucose
HO
O
OH
Halleridone Cornoside
O
OGlc
OCH3O
OHOH O
OGlc
OCH3OOOH
Galioside Hastatoside
Chapter 5 Plant introduction (Part-B)
70
O
OGlc
OHO
OHOH O
OGlc
OCH3OOH
OH
Monotropein Scandoside methyl ester
O
OOH
O
O
O
OHHO O
O
HOOH
OH
OHHO
HO
(-)-2,13-Digalloyl-4-(E)-caffeoyl-L-threonic acid
Chapter 5 Plant introduction (Part-B)
71
(-)-4-(E)-Caffeoyl-L-threonic acid (-)-2-Galloyl-4-(E)-caffeoyl-L-threonic acid
OH
O
O
H
OOH
H
O O
O
HO
HOOH
OH OH
OH
O
HOOH
OH
HO
O
HO
HOHO
HOHO
HO
1, 2, 3, 4, 6-Penta-O-galloyl-β-D-glucose
Chapter 5 Plant introduction (Part-B)
72
3, 3'-Di-O-methylellagic acid 4-(5'-acetyl)-α-L-arabinofuranoside
OH
OHHO OH
O
HO
HHO
H
HOH
H
HO
HO
6-β-Dihydrocornic acid Stenophyllin H1
Chapter 5 Plant introduction (Part-B)
73
Kaempferol Astraglin
Isoquercitin Arjunolicacid
Taraxasterol
Chapter 5 Plant introduction (Part-B)
74
Asiatic acid Tormentic acid
O
O
OO
OCH3
O
O
O
CH3
O
H3CO
O
OO
OCH3
HO
O
O
Cornallegic acid 3'-O-Methyl-3, 4-methylenedioxy ellagic acid
5.1.5. Medicinal and pharmacological properties of the genus Cornus
Several reports about the importance of the genus Cornus in preservation of food and
in traditional medicine have been published.80 One of a mostly grown Cornus specie, C.
officinalis was used in Chinese herbal medicine such as a tonic, analgesic and diuretic
agents.81 The fruits of Cornus species were used to improve the functions of liver and
kidney.82 Anti-bacterial, anti-allergic, anti-microbial, anti-malarial and anti-histamine
activities of many Cornus species are also documented in the literature.83 Many fruits of
Cornus species were studied for their anthocyanin contents. Anthocyanins are a class of
Chapter 5 Plant introduction (Part-B)
75
phenolic compounds that give excellent colours to several vegetables, fruits and having
anticancer, anti-inflammatory, anti-diabetic and antioxidant activities.84-87 In China and
Korea C. controversa was used as a tonic and an astringent.88 It is further reported that the
ethanolic extract of C. mas possess useful activities against Pseudomonas aeruginosa,
Micrococcus luteus and Proteus vulgaris.89 The stones of the fruits have antioxidant
properties90 while C. kousa fruits are edible, attractive and are fermented to wine in some
areas of China where this plant is grownn.91 For immuno-regulatory activity the extract of C.
kousa fruits have been reported in the literature.92,93 The genus Cornus is well known for
tannins particularly hydrolysable tannins and iridoid compounds.94-96 Tannins and related
compounds were documented to have anti-oxidative, anti-microbial,97 anti-cancer,98 and
anti-HIV activities.99
Chapter 6
Results & Discussions
Chapter 6 Results & discussion (Part-B)
77
6.1. Present work
Medicinal and biological uses attributed to the genus Cornus, chemical exploration
of C. macrophylla was carried out in the current study resulted in the isolation and
determination of four new, five reported and three hitherto unreported compounds from C.
macrophylla. Structures of the new compounds were identified by advanced spectroscopic
methods including UV, IR, MS, 1D (1H- and 13C-NMR; BB and DEPT) and 2D NMR (J-
resolved, COSY-45o, NOESY, HSQC, HMBC) experiments. The known compounds were
determined by comparison their spectral data with the reported compounds in the literature.
6.2. New compounds isolated from C. macrophylla
Macrophyllanin A (6)
Chapter 6 Results & discussion (Part-B)
78
Macrophyllanin B (7)
Macrophyllanin C (8)
H3CCOO
O
O
1
4 610
11 18
27
282513
2119
Chapter 6 Results & discussion (Part-B)
79
Macrophyllanin D (9)
6.3. Hitherto unreported compounds from C. macrophylla
Kaempferol (10)
Taraxasterol (11)
Chapter 6 Results & discussion (Part-B)
80
3β- Hydroxy-18α-olean-28-19β-olide (12)
6.4. Reported compounds from C. macrophylla
Betulinic acid (13)
Betulin (14)
Chapter 6 Results & discussion (Part-B)
81
Stigmasterol (15)
Lupeol (16)
Oleanolic acid (17)
Chapter 6 Results & discussion (Part-B)
82
6.2.1. Macrophyllanin A (6)
Compound 6 was obtained as an amorphous white powder. Its molecular
formula was established as C30H46O2 according to the molecular ion peak at m/z
438.3234 a.m.u (calcd. for C30H46O2 438.3230) in the HRESIMS together with the 1H-
and 13C-NMR data.
The IR spectrum for 6 showed an absorption band for lactonic carbonyl group at
1754 cm-1. The 1H-NMR spectrum (Table: 7.1. vide experimental) exhibited seven methyl
signals as singlet in the range of δH 0.85-1.01 ppm and one olefinic proton at δH 5.36 (H-12).
The 13C-NMR spectrum showed resonances for thirty carbon atoms including seven methyl,
ten methylene, five methine and eight quaternary carbons. These characteristic NMR data
suggested that 6 is a pentacyclic triterpene. The 13C-NMR signals at δC 122.4 (C-12) and δC
141.6 (C-13) indicated the presence of a double bond which together with H-18 doublet of
doublet at δH 3.28 (1H, dd, J = 14.3, 4.6 Hz, H-18) led to place the double bond at position
C-12. The downfield chemical shift at δC 86.2 and δC 179.8 showed an oxygen-connected
methine carbon and a carbonyl carbon atom respectively. The 13C-NMR data (Table: 7.1.
vide experimental) of 6 was similar to those of oleanolic acid except the chemical shift of
the oxygen-linked carbon.100 The structure of 6 was further revealed from 2D-NMR spectral
data (Figure: 6.1). The most downfield proton at δH 4.14 (H-21) showed its connectivity
Chapter 6 Results & discussion (Part-B)
83
with the carbon at δC 86.2 (C-21) in the HSQC spectrum. This oxygen-linked carbon was
assigned as C-21 from the HMBC correlations between the proton at δH 4.14 (H-21) and C-
19, C-29, C-30. The HMBC correlation between the proton at δH 4.14 (H-21) and C-28
further confirmed a lactone group between C-21 and C-28. The other part of 6 was
determined to be the same as that of oleanolic acid except the absence of the hydroxyl group
at position C-3. On the basis of spectral evidences compound 6 was established and
characterized as olea-12-en-28, 21 β- olide.
Figure: 6.1: Key HMBC correlations of 6
Chapter 6 Results & discussion (Part-B)
84
6.2.2. Macrophyllanin B (7)
Compound 7 was obtained as a white solid with melting point 244-246 °C. Its
molecular formula was determined as C32H50O4 from the molecular ion peak at m/z
498.7432 a.m.u. (calcd. for C32H50O4 498.7428) in HRESIMS together with the 1H- and
13C-NMR data.
The IR spectrum for 7 exhibited an absorption band for lactonic carbonyl group at
1755 cm-1. The 1H-NMR spectrum (Table: 7.2. vide experimental) exhibited seven methyl
singlets in the range of δH 0.84-1.01 showing the nature of 7 as pentacyclic triterpenoid. The
13C-NMR spectrum showed resonances for thirty carbon atoms including eight methyl, ten
methylene, six methine and eight quaternary carbons. The 1H- and 13C-NMR data (Table:
7.2. vide experimental) of 7 were characteristic of an oleanane skeleton and were in
complete agreement with the proposed structure.101 In addition 1H-NMR showed a singlet at
δH 2.05 (3H) assigned to the methyl of the acetyl group while a doublet of doublet at δH 4.82
(1H, dd, J = 11.8, 4.5 Hz, H-3) was attributed to H-3 proton geminal to the acetyl group. The
13C-NMR spectrum showed the presence of three methine carbon signals at δC 80.4 (C-3),
45.7 (C-18) and δC 85.3 (C-19). The two quaternary carbons resonating at δC 169.6 and δC
178.7 were attributed to the carbonyl of the acetate and lactone group respectively.
H3CCOO
O
O
1
4 610
11 18
27
282513
2119
Chapter 6 Results & discussion (Part-B)
85
The key HMBC correlations of 7 showed cross links between the down field signal
at δH 4.82 (H-3) with the carbonyl of acetyl group and with δC 54.7 (C-5), 25.8 (C-23) and δC
16.4 (C-24). The proton at δH 3.95 (H-19) showed connectivity with the carbon at δC 85.3
(C-19) in the HSQC spectrum. The proton signal at δH 1.76 (H-18) showed correlation with
δC 36.8 (C-14) and δC 30.5 (C-16). The methyl protons resonating at δH 0.74 (3H, H-23)
showed correlations with δC 80.4 (C-3), 37.6 (C-4) and δC 54.7 (C-5). The methyl protons
resonating at δH 0.94 (3H, H-25) showed correlations with δC 38.9 (C-1), 54.7 (C-5) and δC
50.2 (C-9) while the protons resonating at δH 1.01 (3H, H- 26) showed correlations with δC
35.5 (C-7), 41.5 (C-8), 50.2 (C-9) and δC 36.8 (C-14). The methyl protons resonating at δH
0.84 (3H, H-30) showed correlation with δC 85.3 (C-19), 46.2 (C-20), 31.5 (C-21) and δC
22.7 (C-29). Hence the structure of 7 (macrophyllanin B) was elucidated as 3β-
acetoxyolean-28,19β-olide.
Figure: 6.2. Key HMBC correlations of 7
Chapter 6 Results & discussion (Part-B)
86
6.2.3. Macrophyllanin C (8)
Compound 8 was obtained as colorless needles with melting point 272-277 °C. Its
molecular formula was established as C30H46O2 from the molecular ion peak at m/z
438.3435 a.m.u (calcd. for C30H46O2 438.3431) in the HRESIMS together with the 1H-
and 13C-NMR data.
The IR spectrum for 8 showed absorption bands for lactonic carbonyl at 1757cm-1
and C=C bond at 1630 cm-1. The 1H-NMR spectrum (Table: 7.3. vide experimental)
exhibited seven methyl protons signals in the range of δH 0.83-1.01. The 13C-NMR spectrum
showed thirty carbon atoms including seven methyl, ten methylene, five methine and eight
quaternary carbons. These characteristic NMR data suggested that 8 is a pentacyclic
triterpene.
The 1H-NMR spectrum showed five methyl groups at δH 0.85 ( 3H, s, H-25), 1.03
(3H, s, H-26), 0.83 (3H, s, H-27), 0.94 (3H, s, H-29), 0.96 (3H, s, H-30) and two methyl as
doublet at δH 0.96 (3H, d, J = 6.8 Hz, H-23), δH 0.90 (1H, d, J = 6.2 Hz, H-24). The 13C-
NMR signals (Table: 7.3. vide experimental) for two quaternary carbons at δC 136.4, 139.6
Chapter 6 Results & discussion (Part-B)
87
indicated the presence of a double bond located at C-3 (C-5) and signals at δC 86.1 (C-19),
δC 179.9 (C-28) showed an oxygenated carbon and a carbonyl carbon respectively.
The structure of the 8 was further revealed by 2D-NMR spectra (Figure: 6.3). The
proton resonating at δH 3.98 (H-19) showed connectivity with the carbon at δC 86.1 (C-19)
in the HSQC spectrum. The oxygenated carbon was assigned as C-19 due to the HMBC
correlations between the proton at δH 3.98 (H-19) and δC 32.3 (C-21), 23.9 (C-29) and δC
28.7 (C-30). The HMBC correlation between the proton at δH 3.98 (H-19) and the carbon at
δC 179.9 (C-28) supported a lactone group between C-19 and C-28. The methyl protons
resonated at δH 0.96 (3H, H-23) showed correlations with δC 136.4 (C-3), 26.3 (C-4) and δC
139.6 (C-5), while the protons at δH 0.90 (3H, H-24) showed correlations with δC 136.4 (C-
3), 26.3 (C-4), 139.6 (C-5) and δC 21.3 (C-23). The methyl protons at δH 0.84 (3H, H- 25)
showed correlations with δC 42.2 (C-1), 139.6 (C-5) and δC 50.3 (C-9). The methyl proton at
δH 0.94 (3H, H-29) showed correlation with δC 86.1 (C-19), 46.1 (C-20), 32.3 (C-21) and δC
28.7 (C-30). In view of the spectral data 8 (macrophyllanin C) was characterized as A-neo-
18α-olean-3(5)-en-2819β-olide.
O
HHH
H
H
H
O
Figure: 6.3. Key HMBC correlations of 8
Chapter 6 Results & discussion (Part-B)
88
6.2.4. Macrophyllanin D (9)
Compound 9 was assigned the molecular formula C27H36O7 on the basis of molecular
ion peak at m/z 495.2368 [M + Na]+ (calcd. for C27H36O7 472.2253) in the HRESIMS. The IR
spectrum showed absorption bands for OH at 3416 cm−1 and C=O at 1713 cm−1.
O H
OH
OO
1
2 45
7
9
10
12
1415
16
1718
19
20OH
HHH
O O1'2' 2''3'
5'
4' 1''
The 1D-NMR (Table: 7.4. vide experimental) and HSQC spectroscopic data of
compound 9 displayed signals for α, β-unsaturated carbonyl system at δH 7.55 (1H, s, H-1),
159.7 (C-1), 136.4 (C-2) and δC 209.7 (C-3). A trisubstituted double bond was evidenced
from a carbon at δC 142.2 (C-6) and a proton at δH 5.53 (1H, d, J= 5.4 Hz, H-7) and δC 126.4
(C-7). A pair of oxymethylene protons were present at δH 4.03 (2H, s, H-20) and the
corresponding carbon at δC 67.4 (C-20). Four methyl signals were present at δH 1.22 (3H,
s,H-16), δC 16.8 (C-16), δH 1.20 (3H, s,H-17), 23.7 (C-17), δH 0.91 (3H, d, J = 6.4 Hz,H-
18), 14.4 (C-18) and δH 1.73 (3H, s,H-19), δC 10.2 (C-19). Another methylene protons
showed up at δH 2.84 (1H, dd, J = 10.5, 2.5 Hz, H-5a) and 2.15 (1H, dd, J = 10.5, 5.7 Hz, H-
5b). In addition five methines were identified at δH 2.48 (1H, m,H-4) δC 44.2 (C-4),δH 2.38
(1H, s,H-8) δC 42.1 (C-8),δH 3.24 (1H, m, H-10) δC 54.1 (C-10),δH 1.60 (1H, m, H-11)
δC 42.6 (C-11) and δH 1.08 (1H, d, J = 5.3 Hz, H-14), δC 35.6 (C-14).
Chapter 6 Results & discussion (Part-B)
89
The 1H–1H COSY correlations indicated the spin system H-1–H-10, H-7–H-8–H-14
and H-12–H-11–H-18 moieties. NOESY correlations were observed between H-8 and H-4.
These data suggested that 9 possess the tigliane (phorbol) backbone similar to those of the
known compound sapintoxin A.101 Other characteristic resonances included signals
for a tigloyl group and an acetyl moiety. In the HMBC spectrum (Figure: 6.4), H-12 proton
showed a 3J correlation with the carbonyl carbon of the tigloyl group (δC 167.6), confirming
the location of the tigloyl group at δC 76.2 (C-12). Thus, the structure of 9 was determined as
12-O-tiglylphorbol-4-deoxy-4β- phorbol-13- acetate.
Figure: 6.4. Key HMBC correlations of 9
Chapter 6 Results & discussion (Part-B)
90
6.3.1. Kaempferol (10)
The mass spectrum displayed the molecular ion peak at m/z 286.0423 a.m.u.
representing the molecular formula C15H10O6 (calcd. for C15H10O6 286.0419). The 1H-NMR
(Table: 7.5. vide experimental) showed AAˈBBˈ system ortho related proton at δH 7.02 (2H,
d, J = 9 Hz, H-3ˈ, H-5ˈ) and δH 8.13 (2H, d, J = 9 Hz, H-2ˈ, H-6ˈ). The meta related two sets
of doublets was present at δH 6.24 (1H, J = 1.80 Hz, H-6) and δH 6.48 (1H, J = 2.1 Hz, H-8).
The spectral data were similar to the reported compounds in the literature.102
10
6.3.2. Taraxasterol (11)
Compound 11 was isolated as a white powder. HRESIMS showed the [M]+ peak at
m/z 425.3124 a.m.u corresponding to the molecular formula C30H50O (calcd. for C30H50O
425.3120). The 1H- and 13C-NMR (Table: 7.6 vide experimental) of 11 showed seven
methyl signals and thirty carbon signals. The down field region in the 13C-NMR spectrum
showed a quaternary carbon signals at δC 154.8 and germinal methylene carbon signals at δC
107.5. The 1H- and 13C-NMR valves were identical to the taraxasterol reported in the
literature.103
Chapter 6 Results & discussion (Part-B)
91
11
6.3.3. 3β- Hydroxy-18α-olean-28-19β-olide (12)
Compound 12 displayed the [M]+ peak at m/z 456.378 a.m.u corresponding to the
molecular formula C30H48O3 (calcd. for C30H48O3 456.3779). The IR spectrum revealed
absorption bands of a hydroxyl group (3421cm-1) and δ- lactone function at (1739cm-1). The
1H-NMR (Table: 7.7. vide experimental) for 12 represents signals for seven methyls in the
region δH 0.75 to 1.01 showing the nature of triterpenoid. The 1H-NMR showed signal at δH
3.25 (1H, dd, J = 11.5, 5 Hz, H-3) which supported a β-oriented hydroxyl group. In addition,
the13CNMR spectrum showed the presence of one methine carbon signal at δC 77.93 (C-3)
and one carbonyl carbon signal at δC 179.4 (C-28). 1H- and 13C- NMR signals were similar
to the data in the literature.104
Chapter 6 Results & discussion (Part-B)
92
12
6.4.1. Betulinic acid (13)
Compound 13 was isolated as white amorphous powder from the ethyl acatate
fraction of C. macrophylla. Its mass spectrum displayed the [M]+ peak at m/z 456 a.m.u
showing the molecular formula C30H48O3. Its IR spectrum exhibited absorption bands for
OH, C=O, C=C at 3510, 1716 and 1615 cm-1 respectively. The 1H- and 13C-NMR data
(Table: 7.8. vide experimental) were found identical to the reported betulinic acid.105, 106
13
Chapter 6 Results & discussion (Part-B)
93
6.4.2. Betulin (14)
Compound 14 was isolated as a colourless powder. EIMS spectrum showed molecular
ion peak at m/z 442 a.m.u. corresponding to the molecular formula C30H50O2. The IR
spectrum indicated distinctive absorption bands for hydroxyl group at 3448 cm-1 and olefinic
carbons at 1630 cm-1. The 1H- and 13C-NMR spectral data (Table: 7.9. vide experimental)
showed thirty carbons including six methyl, twelve methylene, six methine and six
quaternary carbon atoms. Carbon signals appearing at δC 150.8 (C-20) and δC 109.5 (C-29)
are the prominent peaks for betulin kind of skeleton. A single proton broad doublet at δH
3.19 (br d, J = 3.6 Hz, H-3) and a multiplet at δH 2.32 were assigned to H-3 and H-19
respectively. The spectral data was in correspondence with the reported betulin in the
literature.107
14
Chapter 6 Results & discussion (Part-B)
94
6.4.3. Stigmasterol (15)
Compound 15 was isolated as white needle like crystal. Its mass spectrum showed
the parent molecular ion peak at m/z 412.0123 a.m.u corresponding to the molecular formula
C29H48O (calcd. for C29H48O 412.0119). The 1H-NMR (Table: 7.10. vide experimental) for
compound 15 showed methyl signals from δH 0.85 to 1.06. The 13C-NMR for 15 showed
twenty nine carbon atoms consist of six methyl, nine methylene, eleven methine and three
quaternary carbon signals. The down field region in the 13C-NMR showed a quaternary
carbon signal at δC 141.1 (C-5) and methane carbon signals at δC 121.9 (C-6), 138.4 (C-22)
and δC 129.3 (C-23). The spectral data were identical to the stigmasterol reported in the
literature.108
15
Chapter 6 Results & discussion (Part-B)
95
6.4.4. Lupeol (16)
Lupeol was isolated as white crystals with melting point 209-2110 C. The mass
spectrum exhibited the molecular ion peak at m/z 426.2344 showing to C30H 50O (calcd. for
C30H50O 426.2340). The IR spectrum showed absorption band for hydroxyl group at 3400
cm-1. The 1H- and 13C-NMR spectral data (Table: 7.11. vide experimental) of 16 showed a
pentacyclic triterpinoid of lupane type and after comparison the spectral data with the
reported values confirmed the structure of compound 16 as lupeol.103
16
6.4.5. Oleanolic acid (17)
Compound 17 was isolated as a white amorphous powder. The mass spectrum
showed molecular ion peak at m/z 456.3452 a.m.u corresponding to the molecular formula,
C30H48O3 (calcd. for C30H48O3 456.3448). The 1H-NMR (Table: 7.12. vide experimental)
spectrum of 17 showed seven methyl groups at δH 0.77, 0.79, 0.91, 0.93, 1.21, 0.83 and δH
0.98. A doublet-doublet of one proton at δH 2.83 and a triplet of one vinyl proton at δH 5.24
were attributed to H-18 and H-12 respectively. One methine proton at δH 3.20 (1H, dd, J =
11.2, 4.4 Hz, H-3) represents that 17 has hydroxyl group at position C-3. The spectral data
Chapter 6 Results & discussion (Part-B)
96
(Table: 7.12. vide experimental) were similar to the reported oleanolic acid in the
literature.101
17
6.5. Biological studies
6.5.1. Anti-bacterial assay
The anti-bacterial activity of crude extract and their various fractions along with
their pure isolated compounds were evaluated against four selected bacterial strains;
Proteus mirabilis, Staphylococcus aureus, Escherichia coli and Bacillus cereus (Table:
7.13. vide experimental). The ethyl acetate and methanolic fractions were found to be
active against B. cereus with the inhibitory zone of 13, 14 mm at concentration of (32
μg/mL).
6.5.2. Anti-fungal assay
Various fractions of the stem and aerial parts of C. macrophylla were tested against
five selected fungal strains, Candida albicans, Aspergillus flavus, Microsporum canis,
Fusarium solani and candida glabrata. As results none of them were showed any significant
inhibition. (Table: 7.14. vide experimental)
Chapter 6 Results & discussion (Part-B)
97
6.5.3. Phytotoxicity assay
Various fractions of the stem and aerial parts of C. macrophylla were evaluated for
their in vitro phytotoxic bioassay. All the three fractions of the aerial parts of C.
macrophylla showed significant activities at highest doses against Lemna minor plant.
Acetone extract of the stem of C. macrophylla showed good activity, while MeOH and
MeOH:H2O (1:1) extracts showed moderate activities at highest doses (Table: 7.15. vide
experimental).
6.5.4. Insecticidal assay
The various fractions of the stem and aerial parts of C. macrophylla were evaluated
for the insecticidal assay by using contact toxicity method. All fractions were found to be
inactive at different concentrations (Table: 7.16. vide experimental).
6.5.5 Brine shrimp (Artemia salina) lethality bioassay
Various fractions of the stem and aerial parts of C. macrophylla were evaluated for
brine shrimp lethality bioassay. The n-hexane and EtOAc fractions of the aerial parts of C.
macrophylla showed no cytotoxicity, while methanolic fraction found to be cytotoxic. In the
same way acetone fraction of the stem of C. macrophylla showed no cytoxicity, on the other
hand MeOH and MeOH:H2O (1:1) fractions showed cytotoxicity at highest doses109 (Table:
7.17. vide experimental).
Chapter 7
Experimental
Chapter 7 Experimental (Part-B)
101
7.1. General experimental
The general experimental conditions have already been discussed in chapter 4
(section 4.1.) page no. 35
7.2. Plant Material
The stem bark of C. macrophylla was collected from Bara Gali, Khyber
Pakhtunkhwa, Pakistan in July 2009. The plant was identified by Abdul Majid of the
Department of Botany, Hazara University, Khyber Pakhtunkhwa, Pakistan. A voucher
specimen No. AH-112 was deposited in the herbarium of Hazara University.
7.3 Extraction and isolation
C. macrophylla (6 kg) stem bark was extracted with ethanol at room temperature.
The whole extract was filtered and concentrated under reduced pressure by using rotary
evaporator to obtain a reddish coloured gummy solid (0.5 kg). It was fractionated with
increasing polarity to n-hexane soluble fraction (F1), chloroform soluble fraction (F2) and
ethyl acetate soluble fraction (F3). The two fractions (F1, F2) were not pursued in the
current study.
7.3.1 Fraction of ethyl acetate phase
The ethyl acetate phase was evaporated using rotary evaporator under reduce
pressure and finally the extract dried over anhyd. Na2SO4. The powdery residue (40 g) was
obtained and subjected to column chromatography using silica gel 60 (Merck, 70-230 mm)
eluated with n-hexane and n-hexane_EtOAc solvent in the increasing order of polarity. The
Chapter 7 Experimental (Part-B)
102
eluates were combined on the basis of TLC profile to yield six fractions (AC-1 to AC-6).
Fractions AC-1, AC-3 and AC-4 were not pursued in the present study due to their minute
quantity. Fraction AC-2 was loaded on silica gel column with n-hexane and n-
hexane_EtOAc in increasing order of polarity yielded three fractions ACa, ACb and ACc.
Fraction ACa on prep TLC using n-hexane–EtOAc (7:3) gave three pure compounds, 13
(35 mg), 14 (19 mg), and 16 (21 mg). Fraction ACb following the same procedure and
developing solvent system yielded 2 pure compounds, 17 (28 mg) and 10 (23 mg). Fractions
ACc furnished two pure compounds 11 (31 mg) and 15 (42 mg). Fractions AC-5 was
subjected to column chromatography with n-hexane_EtOAc (4:1) and then chloroform:
methanol (24:1) to afforded two new compounds, 6 (15 mg), 7 (21 mg) and one known
compound, 12 (19 mg) (Scheme: 7.1).
Fraction AC-6 was subjected to silica gel column chromatography, eluted with n-
hexane_EtOAc in the order increasing polarity yielded two fractions AC6a and AC6b.
Fraction AC6a yielded one new compound, 8 (23 mg) on column chromatography and
fraction AC6b yielded another new compound, 9 (17 mg) on silica gel column
chromatography.
Chapter 7 Experimental (Part-B)
103
Scheme: 7.1. Extraction and isolation from C. macrophylla stem.
Percolation with EtOH(three times) at room temp.
n-hexane soluble fractionF1
Aqueous fraction
Solvent removedunder vaccum
Not pursued
Stem bark of Cornus macrophy lla(6 kg)
Ethanolic extract
Solvent removed under vaccum
Solid residue(0.5 kg)
Water suspension
H2O
CHCl3
Aqueous fractionChloroform soluble fractionF2
Not pursued
EtOAc
Aqueous fractionEtOAc soluble fractionF3
Not pursuedPursued
n-hexane
Chapter 7 Experimental (Part-B)
104
Solvent removedunder vaccum
EtOAc soluble fractionF3
Powdered residue(40 g)
Column chromatography
Notpursued
6(15 mg)
AC1 AC2 AC3 AC4 AC5 AC6
Notpursued
i.CCii.TLC
i.CCii.TLC
CC
ACaACb
ACc
AC6a AC6b
CCCC7
(21 mg)
12(19 mg)
8(23 mg)
9(17 mg)
13(35 mg)
16(21 mg)
14(19 mg)
11(31 mg)
15(42 mg)
17(28 mg)
10(23 mg)
Chapter 7 Experimental (Part-B)
105
7.4 Characterization of chemical constituents
Macrophyllanin A (6)
Physical data
Yield: 15 mg
UVλmax ( in CHCl3: 236 (4.73) nm
IR max cm-1: 1742, 1754 (ester/ lactone carbonyls), 1634 (C=C)
HR-ESI-MS m/z : 438.3234 (calcd. for C30H46O2 438.3230)
1H- and 13C-NMR: Table - 7.1.
Table: 7.1. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 6 in CDCl3
Position δH Multiplicity (J in Hz) δC Multiplicity
1a /1b 1.55/1.02 m 39.9 CH2
2a/2b 1.67/1.52 m 18.6 CH2
3a/3b 1.63/1.42 m 42.1 CH2
4 - - 33.6 C
5 0.73 d (7.4) 56.4 CH
6a /6b 1.49/1.30 m 18.2 CH2
Chapter 7 Experimental (Part-B)
106
7a/7b 1.44/1.29 m 33.5 CH2
8 - - 41.5 C
9 1.63 m 50.1 CH
10 - - 38.2 C
11a /11b 1.92/1.91 m 24.5 CH2
12 5.36 t (4.6) 122.4 CH
13 - - 141.6 C
14 - - 42.9 C
15a /15b 2.17/1.19 m 30.2 CH2
16a / 16b 2.11/1.96 m 29.9 CH2
17 - - 44.5 C
18 3.28 dd (14.3, 4.6) 46.6 CH
19a / 19b 1.84/1.33 m 25.4 CH2
20 - - 46.0 C
21 4.14 m 86.2 CH
22a / 22b 2.06/1.83 m 25.5 CH2
23 0.85 s 27.9 CH3
24 0.88 s 15.5 CH3
25 0.90 s 16.5 CH3
26 1.01 s 15.7 CH3
27 0.93 s 13.8 CH3
28 - - 179.8 C
29 0.96 s 23.7 CH3
30 0.89 s 28.7 CH3
Chapter 7 Experimental (Part-B)
107
Macrophyllanin B (7)
Physical data
Yield: 21 mg
UVλmax ( in CHCl3: 244 (4.52) nm
IR max cm-1: 1735, 1755 (ester/ lactone carbonyls)
3054 (C-H, aromatic), 2945(C-H, aliphatic)
HR-EI-MS m/z : 498.7432 (calcd. for C32H50O4 498.7428)
1H- and 13C-NMR: Table- 7.2.
Table: 7.2. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 7 in CDCl3
Position δH Multiplicity (J in Hz) δC Multiplicity
1a/2b 1.79 m 38.9 CH2
2a/2b 1.82 m 35.4 CH2
3 4.82 dd (11.8, 4.5) 80.4 CH
4 - - 37.6 C
5 2.55 m 54.7 CH
6a /6b 1.63/1.30 m 19.2 CH2
7a/7b 1.37/1.29 m 35.5 CH2
8 - - 41.5 C
Chapter 7 Experimental (Part-B)
108
9 2.42 t (4.5) 50.2 CH
10 - - 36.2 C
11a /11b 1.85/1.83 m 24.5 CH2
12a/ 12b 1.35/1.14 m 22.6 CH2
13 2.01 m 34.7 CH
14 - - 36.8 C
15a /15b 2.02/1.13 m 25.6 CH2
16a/ 16b 2.14/1.85 m 30.5 CH2
17 - - 32.6 C
18 1.76 m 45.7 CH
19 3.95 m 85.3 CH
20 - - 46.2 C
21a/ 21b 1.46/1.22 m 31.5 CH2
22a/ 22b 2.03/1.82 m 26.8 CH2
23 0.74 s 25.8 CH3
24 0.72 s 16.4 CH3
25 0.94 s 17.4 CH3
26 1.01 s 16.4 CH3
27 0.95 s 12.7 CH3
28 - - 178.7 C
29 0.91 s 22.7 CH2
30 0.84 s 27.8 CH3
31 2.05 s 21.5 CH3
32 - - 175.7 C
Chapter 7 Experimental (Part-B)
109
Macrophyllanin C (8)
Physical data
Yield: 23 mg
UVλmax ( in CHCl3: 248 (4.74) nm
IR max cm-1: 1730, 1757 (ester/ lactone carbonyls)
3054 (C-H stretching aromatic), 2945(C-H
stretching aliphatic), 1630 (C=C)
HR-ESI-MS : 438.3435 (calcd. for C30H46O2 438.3431)
1H- and 13C-NMR: Table-7.3.
Table: 7.3. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 8 in CDCl3
Position δH Multiplicity (J in Hz) δC Multiplicity
1a/2b 1.82 m 42.2 CH2
2a/2b 1.72/1.83 m 37.3 CH2
3 - - 136.4 C
4 2.62 m 26.3 CH
5 - - 139.6 C
6a /6b 1.58/1.35 m 19.6 CH2
7a/7b 1.32/1.24 m 32.4 CH2
Chapter 7 Experimental (Part-B)
110
8 - - 40.7 C
9 2.51 m 50.3 CH
10 - - 49.7 C
11a /11b 1.82/1.78 m 23.5 CH2
12a / 12b 1.29/1.09 m 26.5 CH2
13 2.02 m 36.4 CH
14 - - 39.7 C
15a /15b 2.09/ 2.23 m 28.2 CH2
16a / 16b 2.09/1.80 m 31.9 CH2
17 - - - C
18 1.82 s 46.7 CH
19 3.98 s 86.1 CH
20 - - - C
21a / 21b 1.45/1.23 m 32.3 CH2
22a / 22b 2.02/1.83 m 25.2 CH2
23 0.96 d (6.8) 21.3 CH3
24 0.90 d (6.2) 16.4 CH3
25 0.85 s 19.1 CH3
26 1.03 s 14.0 CH3
27 0.83 s 13.5 CH3
28 - - 179.9 C
29 0.94 s 23.9 CH3
30 0.96 s 28.7 CH3
Chapter 7 Experimental (Part-B)
111
Macrophyllanin D (9)
Physical data
Yield: 17 mg
UVλmax ( in CHCl3: 205 (4.05) nm
IR max cm-1: 3416 (hydroxyl), 1713 (carbonyl group)
2945(C-H stretching aliphatic), 1630 (double bond)
HR-ESI-MS: 495.2368 [M + Na]+ (calcd. for C27H36O7 472.2253)
1H- and 13C-NMR: Table-7.4.
Table: 7.4. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 9 in CDCl3
Position δH Multiplicity (J in Hz) δC Multiplicity
1 7.55 s 159.7 CH
2 - - 136.4 C
3 - - 209.7 C
4 2.48 m 44.2 CH
5a/5b 2.84/2.15 dd (10.5, 2.5)/dd (10.5, 5.7) 29.6 CH2
6 - - 142.2 C
7 5.53 d (5.4) 126.4 CH
Chapter 7 Experimental (Part-B)
112
8 2.38 m 42.1 CH
9 - - 77.8 C
10 3.24 m 54.1 CH
11 1.60 m 42.6 CH
12 5.47 d (9.7) 76.2 CH
13 - - 65.4 C
14 1.08 d (5.3) 35.6 CH
15 - - 25.6 C
16 1.22 s 16.8 CH3
17 1.20 s 23.7 CH3
18 0.91 d (6.4) 14.4 CH3
19 1.73 s 10.2 CH3
20a/20b 4.03/3.99 s 67.4 CH2
1′ - - 167.6 C
2′ - - 128.5 C
3′ 6.84 m 137.4 CH
4′ 1.80 d (7.0 ) 14.4 CH3
5′ 1.84 s 12.2 CH3
1" - - 179.4 C
2" 2.10 s 23.4 CH3
Chapter 7 Experimental (Part-B)
113
Kaempferol (10)
Physical data
Yield: 23 mg
UV max (MeOH): 265 (3.62) nm
IR max cm-1: 3400 (hydroxyl), 2944 (C–H), 1458 (C=C)
HR-ESI-MS m/z : 286.0423 (calcd. for C15H10O6 286.0419)
1H- and 13C-NMR: Table -7.5.
Table: 7.5. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 10 in CDCl3
Position δH Multiplicity (J in Hz) δC Multiplicity
1 - - -
2 - - 147.1 C
3 - - 136.2 C
4 - - 175.8 C
5 - - 161.5 C
6 6.24 d (1.80) 98.6 CH
7 - - 164.3 C
8 6.48 d (2.1) 93.8 CH
9 - - 157.2 C
Chapter 7 Experimental (Part-B)
114
10 - - 103.4 C
1ˈ - - 122.4 C
2ˈ 8.13 d (9.1) 129.8 CH
3ˈ 7.02 d (9.1) 115.6 CH
4ˈ - - 159.5 C
5ˈ 7.02 d (9.2) 115.6 CH
6ˈ 8.13 d (9.2) 129.7 CH
Taraxasterol (11)
Physical data
Yield: 31 mg
UV max (MeOH): 237 (3.55) nm
IR max cm-1: 3445 (hydroxyl), 1615 (C=C)
HR-ESI-MS m/z : 425.3124 [M - H ]+ (calcd. for C30H50O 425.3120)
1H- and 13C-NMR: Table-7.6.
Chapter 7 Experimental (Part-B)
115
Table: 7.6. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 11 in CDCl3
Position δH Multiplicity (J in Hz) δC Multiplicity
1a /1b 1.70/0.95 m 38.7 CH2
2 1.60 m 37.3 CH2
3 3.15 m 79.1 CH
4 - - 38.6 C
5 0.71 m 55.4 CH
6a /6b 1.53/1.37 m 18.6 CH2
7a/7b 1.38 m 34.2 CH2
8 - - 40.2 C
9 1.31 s 50.4 CH
10 - - 37.2 C
11a /11b 1.53/1.27 m 23.7 CH2
12a / 12b 1.67/1.12 m 26.2 CH2
13 1.58 m 39.1 CH
14 - - 42.1 C
15a /15b 1.67/.96 m 26.6 CH2
16a / 16b 1.22/1.15 m 38.2 CH2
17 - - 34.4 C
18 0.95 m 48.6 CH
19 2.34 m 39.5 CH
20 - - 154.8 C
21a/21b 2.40/2.18 m 25.4 CH2
22 1.36 m 38.7 CH2
23 0.97 s 28.1 CH3
24 0.78 s 15.3 CH3
25 0.90 s 16.2 CH3
26 0.98 s 15.9 CH3
27 0.92 s 14.7 CH3
Chapter 7 Experimental (Part-B)
116
28 0.85 s 19.5 CH3
29 1.01 d (7.1) 25.5 CH3
30a/30b 4.61/4.58 s 107.5 CH2
3β- Hydroxy-18α-olean-28-19β-olide (12)
Physical data
Yield: 19 mg
UV max (MeOH): 247 (4.35) nm
IR max cm-1: 3421 (hydroxyl), 1739 (lactone carbonyls)
HR-ESI-MS m/z : 456.3783 (calcd. for C30H48O3 456.3779)
1H- and 13C-NMR: Table- 7.7.
Chapter 7 Experimental (Part-B)
117
Table: 7.7. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 12 in CDCl3
Position δH Multiplicity (J in Hz) δC Multiplicity
1a /1b 1.57/1.03 m 38.9 CH2
2a/2b 2.32/1.88 m 27.33 CH2
3 3.25 dd ( 11.5, 5) 77.93 CH
4 - - 38.8 C
5 0.74 d, (11.9) 55.4 CH
6a /6b 1.50/1.31 m 18.1 CH2
7a /7b 1.45/1.30 m 33.6 CH2
8 - - 40.5 C
9 1.64 d (2.6) 51.2 CH
10 - - 37.2 C
11a /11b 1.91/1.91 m 26.5 CH2
12a / 12b 1.66/1.11 m 20.6 CH2
13 1.57 m 35.9 CH
14 - - 39.9 C
15a /15b 2.18/1.20 m 27.8 CH2
16a / 16b 2.12/1.97 m 31.9 CH2
17 - - 33.5 C
18 1.76 m 46.6 CH
19 3.05 m 86.0 CH
20 - - 46.0 C
21a / 21b 1.48/1.24 m 32.2 CH2
22a / 22b 2.06/1.83 m 25.5 CH2
23 0.77 s 27.9 CH3
24 0.75 s 15.3 CH3
25 0.94 s 16.5 CH3
26 1.01 s 15.5 CH3
27 0.96 s 13.6 CH3
28 0.83 - 179.4 C
Chapter 7 Experimental (Part-B)
118
29 0.90 s 23.9 CH3
30 0.86 s 28.7 CH3
Betulinic acid (13)
Physical data
Yield: 35 mg
UV max (MeOH) in nm: 241 (4.22) nm
IR max cm-1: 3510 (hydroxyl), 1716 (carbonyl group), 1615 (C=C).
EI-MS m/z (rel.int. %): 456 [M]+ (37), 438 (10), 411 (7), 248 (38),
234 (25), 207 (53), and 189 (100).
1H- and 13C-NMR: Table-7.8.
Chapter 7 Experimental (Part-B)
119
Table: 7.8. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 13 in CDCl3
Position δH Multiplicity (J in Hz) δC Multiplicity
1a /1b 1.51 m 38.5 CH2
1a /1b 1.29 m 27.8 CH2
3 3.30 dd, (10.4, 3.9) 78.6 CH
4 - - 37.7 C
5 1.44 m 55.6 CH
6a/6b 1.56 m 18.6 CH2
7a /7b 1.49 m 34.5 CH2
8 - - 40.8 C
9 1.37 m 50.3 CH
10 - - 37.1 C
11a/11b 1.42 m 20.9 CH2
12a/12b 1.77 m 25.6 CH2
13 1.40 m 37.3 CH
14 - - 42.6 C
15a/15b 1.79 m 30.8 CH2
16a/16b 1.45 m 32.4 CH2
17 - - 56.5 C
18 1.79 m 46.7 CH
19 3.04 br s 49.2 CH
20 - - 150.5 C
21a/21b 1.45 m 29.5 CH2
22a/22b 1.76 m 32.1 CH2
23 0.80 s 27.7 CH3
24 0.83 s 15.6 CH3
25 0.79 s 16.4 CH3
26 0.87 s 16.6 CH3
27 1.15 s 14.7 CH3
28 - - 180.5 C
Chapter 7 Experimental (Part-B)
120
29a/29b 4.56/4.43 m 109.8 CH2
30 1.55 s 19.37 CH3
Betulin (14)
Physical data
Yield: 19 mg
UV max (CHCl3): 244 (4.1) nm
IR max : 3448 (hydroxyl), 1630 (C=C)
EI-MS m/z (rel. int. %): 442 [M]+ (30) 412 (6), 234 (23), 220 (27), 207 (50), 189
(100), 175 (32)
1H- and 13C-NMR: Table-7.9.
Table: 7.9. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 14 in CDCl3
Position δH Multiplicity (J in Hz) δC Multiplicity
1a/1b 1.41 m 38.9 CH2
2a/2b 1.60 m 20.7 CH2
3 3.19 dd (11.2, 3.6) 78.8 CH
4 - - 38.6 C
5 1.39 m 54.9 CH
Chapter 7 Experimental (Part-B)
121
6a/6b 1.52 m 17.8 CH2
7a/7b 1.47 m 33.9 CH2
8 - - 40.7 C
9 1.36 m 54.8 CH
10 - - 36.9 C
11a/11b 1.46 m 27.7 CH2
12a/12b 1.57 m 24.8 CH2
13 1.39 m 36.9 CH
14 - - 42.4 C
15a/15b 1.59 m 26.9 CH2
16a/15b 1.34 m 28.8 CH2
17 - - 45.9 C
18 1.71 m 49.8 CH
19 2.32 m 48.6 CH
20 - - 150.8 C
21a/21b 1.55 m 29.3 CH2
22a/22b 1.53 m 34.5 CH2
23 0.93 s 27.7 CH3
24 0.79 s 15.8 CH3
25 0.73 s 15.7 CH3
26 0.95 s 14.8 CH3
27 1.01 s 14.5 CH3
28 3.31 m 60.6 CH2
29a/29b 4.64/4.55 s 109.5 CH2
30 1.82 s 19.7 CH3
Chapter 7 Experimental (Part-B)
122
Stigmasterol (15)
HO
H
H
H
H
Physical data
Yield: 42 mg
UV max (MeOH) in nm: 235 (4.53) nm
IR max cm-1: 3475 (hydroxyl), 1615 (C=C)
HR-ESI-MS m/z : 412.0123 (calcd. for C29H48O 412.0119)
1H- and 13C-NMR: Table -7.10.
Table: 7.10. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 15 in CDCl3
Position δH Multiplicity (J in Hz) δC Multiplicity
1a /1b 1.72/0.94 m 37.4 CH2
2a/2b 1.61 m 31.8 CH2
3 3.49 dd, (10.5, 4.2) 72.1 CH
4a/4b - - 42.4 CH2
5 0.72 m 141.1 C
6a /6b 5.12 m 121.9 CH2
7a/7b 1.37 m 31.8 CH2
8 - - 32.1 C
Chapter 7 Experimental (Part-B)
123
9 1.33 m 50.3 CH
10 - - 36.8 C
11a /11b 1.52/1.26 m 21.2 CH2
12a / 12b 1.66/1.11 m 39.8 CH2
13 1.57 m 42.4 C
14 - - 57.1 CH
15a /15b 1.65/.95 m 24.4 CH2
16a / 16b 1.21/1.14 m 28.5 CH2
17 - - 56.3 CH
18 1.06 m 12.2 CH
19 1.25 d 19.5 CH3
20 1.03 m 40.6 CH
21 0.92 m 21.1 CH
22 4.61 m 138.4 CH
23a/23b 4.60 s 129.3 CH2
24 0.77 t (7.1) 51.4 CH3
25 0.90 s 32.1 CH
26 1.06 d ( 6.6) 21.2 CH3
27 1.01 d ( 6.6) 21.1 CH3
28 0.85 s 25.5 CH3
29 0.98 s 12.4 CH3
Chapter 7 Experimental (Part-B)
124
Lupeol (16)
Physical data
Yield: 21 mg
UV max (MeOH): 210 (4.53) nm
IR max cm-1: 3400 (hydroxyl), 2944 (C–H), 1458 (C=C)
HR-ESI-MS m/z : 426.2344 (calcd. for C30H50O 426.2340)
1H- and 13C-NMR: Table- 7.11.
Table: 7.11. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 16 in CDCl3
Position δH Multiplicity (J in Hz) δC Multiplicity
1a /1b 1.63 /0.94 m 38.9 CH2
2a/2b 1.60 m 20.7 CH2
3 3.18 dd (9.6, 6.2) 78.8 CH
4 - - 38.6 C
5 0.69 m 54.9 CH
6a /6b 1.51/1.38 m 17.8 CH2
7a/7b 1.41 m 33.9 CH2
8 - - 40.7 C
Chapter 7 Experimental (Part-B)
125
9 1.33 m 54.8 CH
10 - - 36.9 C
11a /11b 1.35 m 27.7 CH2
12a /12b 1.69 /1.09 m 24.8 CH2
13 1.56 m 36.9 CH
14 - - 42.4 C
15a /15b 1.56/0.95 m 26.9 CH2
16a/16b 1.46 m 28.8 CH2
17 - - 45.9 C
18 1.56 m 49.8 CH
19 2.34 m 48.6 CH
20 - - 150.8 C
21a/21b 1.25 m 29.3 CH2
22a/22b 1.20 m 34.5 CH2
23 0.94 s 27.7 CH3
24 0.76 s 15.8 CH3
25 0.83 s 15.7 CH3
26 0.98 s 14.8 CH3
27 0.94 s 14.5 CH3
28 0.81 s 60.6 CH3
29a/29b 4.64/4.55 s 109.5 CH2
30 1.72 s 19.7 CH3
Chapter 7 Experimental (Part-B)
126
Oleanolic acid (17)
Physical data
Yield: 28 mg
UV max (MeOH): 254 (4.34) nm
IR max cm-1: 3423 (hydroxyl), 1615 (C=C), 1706 (C=O)
HR-ESI-MS m/z : 456.3452 (calcd. for C30H48O3 456.3448)
1H- and 13C-NMR: Table- 7.12.
Table: 7.12. 1H- (600 MHz), 13C-NMR (100 MHz) NMR data for 17 in CDCl3
Position δH Multiplicity (J in Hz) δC Multiplicity
1a /1b 1.61/0.93 m 38.9 CH2
2a/2b 1.62 m 26.7 CH2
3 3.20 dd (11.2, 4.4) 78.6 CH
4 - - 38.6 C
5 0.72 m 54.7 CH
6a /6b 1.531.36 m 18.8 CH2
7a/7b 1.43 m 33.1 CH2
8 - - 40.3 C
9 1.32 m 49.8 CH
10 - - 36.6 C
Chapter 7 Experimental (Part-B)
127
11a /11b 1.35 m 23.7 CH2
12 5.24 t (3.5) 122.4 CH
13 1.55 m 143.5 C
14 - - 42.3 C
15a /15b 1.54/.94 m 26.8 CH2
16a/16b 1.45 m 23.5 CH2
17 - - 45.2 C
18 2.83 dd (13.2, 3.6) 41.4 CH
19a/19b 2.35 m 45.6 CH2
20 - - 30.4 C
21a/21b 1.24 m 34.5 CH2
22a/22b 1.21 m 32.4 CH2
23 0.77 s 27.9 CH3
24 0.79 s 15.8 CH3
25 0.91 s 15.4 CH3
26 0.93 s 16.7 CH3
27 1.21 s 26.2 CH3
28 0.83 - 182.8 C
29 0.98 s 33.2 CH3
30 1.01 s 23.4 CH3
Chapter 7 Experimental (Part-B)
128
Table: 7.13. Anti-bacterial activity of C. macrophylla
Sample P. m S. a E .c B. c
F1 12 X x X
F2 x 11 x X
F3 12 X x 13
F4 8 10 x 14
F5 10 11 x 8
Betulin (13) x X x X
Betulinic acid (14) x X x X
DMSO (Negative Control) x X x X
Imipenem 10µg/Disc (Positive
Control)
28 23 34 32
Keywords: F1= n- hexane; F2= CHCl3; F3= EtOAc; F4= MeOH; F5= crude extract.
P. m= Proteus merablus; S. a= Staphylococcus aureus; E. c= Escherichia coli; B.
c= Bacillus cereus
Table: 7.14. Anti-fungal assay activity of C. macrophylla
Fractions/Extracts Name of Fungus % inhibition Std. Drug Mic (µg/mL)
F1
C. a 0 Miconazole (110.8)A. f 0 Amphotericin B (20.20)M. c 20 Miconazole (98.4)F. s 15 Miconazole (73.25)C. g 0 Miconazole (110.8)
F2
C. a 0 Miconazole (110.8)A. f 15 Amphotericin B (20.20)M. c 15 Miconazole (98.4)F. s 20 Miconazole (73.25)C. g 0 Miconazole (110.8)
F3
C. a 0 Miconazole (110.8)A. f 10 Amphotericin B (20.20)M. c 15 Miconazole (98.4)F. s 15 Miconazole (73.25)C. g 0 Miconazole (110.8)
A1C. a 0 Miconazole (110.8)A. f 05 Amphotericin B (20.20)
Chapter 7 Experimental (Part-B)
129
Keywords: Aerial parts = F, stem = A, F1 = n-hexane, F2 = EtOAc, F3 = MeOH, A1 =
Acetone, A2 = Methanol, A3 = MeOH:H2O (1:1), C. a = Candida albicans, A. f = Aspergillus
flavus, M. c = Microsporum canis, F. s = Fusarium solani, C. g= Candida glabrata
(Table: 7.15.: Phytotoxicity assay of C. macrophylla
Extract/Fractions
CompoundsConc.
(µg/mL)
No. of fronds% Growthregulation
Std. DrugConc.
(µg/mL)Samples Control
F11000 06
20
70
0.015100 14 3010 18 10
F21000 06
20
700.015100 16 20
10 18 10
F3
1000 06
20
70
0.015100 14 30
10 18 10
A1
1000 10
20
50
0.015100 12 40
10 17 15
A2
1000 11
20
45
0.015100 16 20
10 18 10
M. c 25 Miconazole (98.4)F. s 20 Miconazole (73.25)C. g 0 Miconazole (110.8)
A2
C. a 0 Miconazole (110.8)A. f 10 Amphotericin B (20.20)M. c 30 Miconazole (98.4)F. s 20 Miconazole (73.25)C. g 0 Miconazole (110.8)
A3
C. a 0 Miconazole (110.8)A. f 0 Amphotericin B (20.20)M. c 15 Miconazole (98.4)F. s 15 Miconazole (73.25)C. g 0 Miconazole (110.8)
Chapter 7 Experimental (Part-B)
130
A3
1000 12
20
40
0.015100 16 20
10 18 10
Keywords: Aerial parts = F, stem = A, F1 = n-hexane, F2 = EtOAc, F3 = MeOH,
A1 = Acetone, A2 = Methanol, A3 = MeOH: H2O (1:1)
Table: 7.16. Insecticidal activity of C. macrophylla
Fractions Name of Insects % Mortality
F1Tribolium castaneum 0Rhyzopertha dominica 0Callosbruchus analis 20
F2Tribolium castaneum 0Rhyzopertha dominica 0Callosbruchus analis 0
F3Tribolium castaneum 0Rhyzopertha dominica 0Callosbruchus analis 0
A1Tribolium castaneum 0Rhyzopertha dominica 20Callosbruchus analis 0
A2Tribolium castaneum 0Rhyzopertha dominica 0Callosbruchus analis 0
A3Tribolium castaneum 0Rhyzopertha dominica 0Callosbruchus analis 20
Keywords: Aerial parts = F, stem = A, F1 = n-hexane, F2 = EtOAc, F3 = MeOH, A1 =
Acetone, A2 = Methanol, A3 = MeOH: H2O (1:1)
Chapter 7 Experimental (Part-B)
131
Table: 7.17. Brine Shrimp activity of C. macrophylla
FractionsDoses
(µg/mL)No. of
shrimpsNo. of
survivorsLD50
(µg/mL) STD. DrugLD50
(µg/mL)
F11000 30 22
413892.80 Etoposide 7.4625100 30 2510 30 26
F21000 30 25
4251653 Etoposide 7.4625100 30 2710 30 28
F31000 30 16
1050.50 Etoposide 7.4625100 30 2110 30 28
A11000 29 19
1039111.1 Etoposide 7.4625100 29 2210 29 23
A21000 29 15
981.641 Etoposide 7.4625100 29 2210 29 20
A31000 29 13
607.64 Etoposide 7.4625100 29 2210 29 28
Keywords: Aerial parts = F, stem = A, F1 = n-hexane, F2 = EtOAc, F3 = MeOH,
A1 = Acetone, A2 = Methanol, A3 = MeOH: H2O (1:1)
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List of Publication
140
1. Ghias Uddin, Ashfaq Ahmad Khan, Muhammad Alamzeb, Saqib Ali, Mamoon-Ur-
Rashid, Anwar Sadat, Muhammad Alam, Abdul Rauf and Wali Ullah. Biological
screening of ethyl acetate extract of Hedera nepalensis stem. African Journal of
Pharmacy and Pharmacology Vol. 6(42), pp. 2934-2937, 15 November, 2012 Available
online at http://www.academicjournals.org/AJPP, DOI: 10.5897/AJPP12.828. ISSN
1996-0816 © 2012 Academic Journals.
2. Muhammad Alamzeb, M. Rafiullah Khan, Saqib Ali, Syed Qaiser Shah, Mamoon-Ur-
Rashid, Ashfaq Ahmad Khan. Bioassay guided isolation and characterization of anti-
microbial and anti-trypanosomal agents from Berberis glaucocarpa Stapf. African
Journal of Pharmacy and Pharmacology /23.01.13/3444. OPEN ACCESS JOURNALS.
http://www.academicjournals.org/AJPP.
3. Ghias Uddin, Waliullah, Abdur Rauf, Bina Shaheen Siddique, Ashfaq Ahmed, Chand
Bibi, Muhammad Qaiser and Sadiq Azam. Phytochemical Screening and Antimicrobial
activity of Cornus macrophylla. Middle-East Journal of Scientific Research 9(4); 516-
519,2011 ISSN 1990-9233 IDOSI publication, 2011
4. Ghias Uddin, Waliullah, Bina Shaheen Siddiqui, Muhammad Alam, Anwar Sadat,
Ashfaq Ahmed and AlaUddin "Chemical Constituents and Biological Screening of
Grewia optiva Drummond ex Burret Whole Plant." American-Eurasian J Agric &
Environ Sci 11.4 (2011): 542-546.
5. Muhammad Alam, Ghias Uddin, Anwar Sadat, Naveed muhammd, Ashfaq Ahmad
Khan and Bina S. Siddiqui "Evaluation of Viburnum grandiflorum for its in-vitro
pharmacological screening." African Journal of Pharmacy and Pharmacology6.22
(2012): 1606-1610.
List of Publication
141
6. Ghias Uddin , Ashfaq A Khan , Anwar Sadat , Igoli O John , Dima Semaan , Carol
Clements, Bina S. Siddiqui, Valerie A. Ferro, Alexander I. Gray
Antidiabetic and Antimicrobial potential of pentacyclic terpenoids from Cornus
macrophylla Wall. ex Roxb. (Submitted)
7. Muhammad Alamzeb,*,† Ajmal Khan,‡ Qaiser Jamal,*, ‡ Syed Akram Shah,‡ M. Rafiullah
Khan,§ Saqib Ali,┴ Mamoon-Ur-Rashid,§ Ashfaq Ahmad Khan§
Novel and highly active Anti-Leishmanial agents against Leishmanial clinical field
isolates KHW23 from Berberis glaucocarpa Stapf. (Submitted)
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